Bispecific antibodies against CD3 and BCMA

ABSTRACT

A bispecific antibody specifically binding to the two targets human CD3ε and human BCMA, wherein the variable domains of one antibody portion are replaced by each other, and characterized in that the binding of said antibody is not reduced by APRIL and not reduced by BAFF, said antibody does not alter APRIL-dependent NF-κB activation, BAFF-dependent NF-κB activation, and does not alter NF-κB activation without BAFF and APRIL is useful as a therapeutic agent.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:3681_0010003_SL.txt; Size: 36,997 bytes; and Date of Creation: Jul. 27,2015) is herein incorporated by reference in its entirety.

The present invention relates to novel bispecific antibodies againstCD3ε and BCMA, their manufacture and use.

BACKGROUND OF THE INVENTION

Human B cell maturation target, also known as BCMA; TR17_HUMAN, TNFRSF17(UniProt Q02223), is a member of the tumor necrosis receptor superfamilythat is preferentially expressed in differentiated plasma cells [Laabiet al. 1992; Madry et al. 1998]. BCMA is a non glycosylated type IIItransmembrane protein, which is involved in B cell maturation, growthand survival. BCMA is a receptor for two ligands of the TNF superfamily:APRIL (a proliferation-inducing ligand), the high-affinity ligand toBCMA and the B cell activation factor BAFF, the low-affinity ligand toBCMA (THANK, BlyS, B lymphocyte stimulator, TALL-1 and zTNF4). APRIL andBAFF show structural similarity and overlapping yet distinct receptorbinding specificity. The negative regulator TACI also binds to both BAFFand APRIL. The coordinate binding of APRIL and BAFF to BCMA and/or TACIactivates transcription factor NF-κB and increases the expression ofpro-survival Bcl-2 family members (e.g. Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1)and the downregulation of pro-apoptotic factors (e.g. Bid, Bad, Bik,Bim, etc.), thus inhibiting apoptosis and promoting survival. Thiscombined action promotes B cell differentiation, proliferation, survivaland antibody production (as reviewed in Rickert R C et al., Immunol Rev(2011) 244 (1): 115-133). Antibodies against BCMA are described e.g. inGras M-P. et al. Int Immunol. 7 (1995) 1093-1106, WO200124811, andWO200124812. The use of anti-BCMA antibodies for the treatment oflymphomas and multiple myeloma are mentioned e.g. in WO2002066516 andWO2010104949.

The TCR/CD3 complex of T-lymphocytes consists of either a TCR alpha(α)/beta (β) or TCR gamma (γ)/delta (δ) heterodimer coexpressed at thecell surface with the invariant subunits of CD3 labeled gamma (γ), delta(δ), epsilon (ε), zeta (ζ) and eta (η). Human CD3ε is described underUniProt P07766 (CD3E_HUMAN). An anti CD3ε antibody described in thestate of the art is SP34 (Yang S J, The Journal of Immunology (1986)137; 1097-1100). SP34 reacts with both primate and human CD3. SP34 isavailable from Pharmingen. A further anti CD3 antibody described in thestate of the art is UCHT-1 (see WO2000041474). A further anti CD3antibody described in the state of the art is BC-3 (Fred HutchinsonCancer Research Institute; used in Phase I/II trials of GvHD, Anasettiet al., Transplantation 54: 844 (1992)). SP34 differs from UCHT-1 andBC-3 in that SP-34 recognizes an epitope present on solely the ε chainof CD3 (see Salmeron et al., (1991) J. Immunol. 147: 3047) whereasUCHT-1 and BC-3 recognize an epitope contributed by both the ε and γchains. The sequence of an antibody with the same sequence as ofantibody SP34 is mentioned in WO2008119565, WO2008119566, WO2008119567,WO2010037836, WO2010037837 and WO2010037838. A sequence which is 96%identical to VH of antibody SP34 is mentioned in U.S. Pat. No. 8,236,308(WO2007042261). VH and VL sequences of a further antibody with the samesequences as of SP34 are shown in SEQ ID NO:7 and 8.

A wide variety of recombinant bispecific antibody formats have beendeveloped in the recent past, e.g. by fusion of, e.g. an IgG antibodyformat and single chain domains (see Kontermann R E, mAbs 4:2, (2012)1-16). Bispecific antibodies wherein the variable domains VL and VH orthe constant domains CL and CH1 are replaced by each other are describedin WO2009080251 and WO2009080252.

An approach to circumvent the problem of mispaired byproducts, which isknown as ‘knobs-into-holes’, aims at forcing the pairing of twodifferent antibody heavy chains by introducing mutations into the CH3domains to modify the contact interface. On one chain bulky amino acidswere replaced by amino acids with short side chains to create a ‘hole’.Conversely, amino acids with large side chains were introduced into theother CH3 domain, to create a ‘knob’. By coexpressing these two heavychains (and two identical light chains, which have to be appropriate forboth heavy chains), high yields of heterodimer formation (‘knob-hole’)versus homodimer formation (‘hole-hole’ or ‘knob-knob’) was observed(Ridgway J B, Presta L G, Carter P; and WO1996027011). The percentage ofheterodimer could be further increased by remodeling the interactionsurfaces of the two CH3 domains using a phage display approach and theintroduction of a disulfide bridge to stabilize the heterodimers(Merchant A. M, et al, Nature Biotech 16 (1998) 677-681; Atwell S,Ridgway J B, Wells J A, Carter P., J MoI Biol 270 (1997) 26-35). Newapproaches for the knobs-into-holes technology are described in e.g. inEP 1870459A1. Although this format appears very attractive, no datadescribing progression towards the clinic are currently available. Oneimportant constraint of this strategy is that the light chains of thetwo parent antibodies have to be identical to prevent mispairing andformation of inactive molecules. Thus this technique is not appropriatefor easily developing recombinant, bispecific antibodies against twotargets starting from two antibodies against the first and the secondtarget, as either the heavy chains of these antibodies and/or theidentical light chains have to be optimized. Xie, Z., et al, J ImmunolMethods 286 (2005) 95-101 refers to a format of bispecific antibodyusing scFvs in combination with knobs-into-holes technology for the FCpart. WO 2012116927 and WO 2010/145792, and mention exchanging the CH1and CL domains. WO 2009/080254 mentions knob in hole constructs forproducing bispecific antibodies.

Antibodies against BCMA are described e.g. in Gras M-P. et al. IntImmunol. 7 (1995) 1093-1106, WO200124811, WO200124812, WO2010104949 andWO2012163805. Antibodies against BCMA and their use for the treatment oflymphomas and multiple myeloma are mentioned e.g. in WO2002066516 andWO2010104949. WO2013154760 relates to chimeric antigen receptorscomprising a BCMA recognition moiety and a T-cell activation moiety.

Ryan, M C et al., Mol. Cancer Ther. 6 (2007) 3009-3018 relateselectively targeting of BCMA for plasma cell malignancies. AntibodySG1, with ligand blocking activity could promote cytotoxicity ofmultiple myeloma (MM) cell lines as naked antibodies or as antibody-drugconjugates (ADC). SG1, an inhibitory BCMA antibody, blocksAPRIL-dependent activation of nuclear factor-κB in a dose-dependentmanner in vitro. Cytotoxicity of SG1 was assessed as a naked antibodyafter chimerization with and without Fc mutations that enhance FcγRIIIAbinding. Ryan also mentions antibody SG2 which does not significantlyinhibit APRIL binding to BCMA. However SG2 showed a 20 fold higher IC₅₀value as SG1 measured as cytotoxic activity of a drug conjugate againstBCMA positive myeloma cell lines. Ryan conclude that BCMA antibodies canact on MM cell lines through multiple mechanisms that include inhibitionof APRIL-dependent NF-κB activation, promotion of tumor cell lysis bynatural killer cell-mediated ADCC activity, and induction ofcytotoxicity by ADCs.

Bispecific antibodies against CD3 and BCMA are mentioned inWO2007117600, WO2009132058, WO2012066058, and WO2012143498.

SUMMARY OF THE INVENTION

The invention comprises a bispecific antibody specifically binding tothe two targets human CD3ε (further named also as “CD3”) and theextracellular domain of human BCMA (further named also as “BCMA”),characterized in that the binding of said antibody in a concentration of6.25 nM is not reduced by 140 ng/ml murine APRIL for more than 10%,preferably not reduced by for more than 1% measured in an ELISA assay asOD at 450 nm compared to the binding of said antibody to human BCMAwithout APRIL. Preferably the bispecific antibody is characterized inthat the binding of said antibody in a concentration of 50 nM is notreduced by 140 ng/ml murine APRIL for more than 10%, measured in anELISA assay as OD at 450 nm, compared to the binding of said antibody tohuman BCMA without APRIL.

Preferably the antibody according to the invention is characterized inshowing an EC50 value for binding of anti-BCMA antibodies to H929 cells(ATCC® CRL9068™) of 15 nM or lower.

The invention relates to a bispecific antibody specifically binding tothe two targets human CD3ε (further named also as “CD3”) and theextracellular domain of human BCMA (further named also as “BCMA”),characterized in that

-   -   i) said bispecific antibody is a fusion protein of a Fab        fragment of an anti-CD3 antibody chemically linked at its        N-terminus to the C-terminus of a Fab fragment of an anti-BCMA        antibody and    -   ii) the variable domains VL and VH of the anti-CD3 antibody or        the constant domains CL and CH1 are replaced by each other.

Preferably a VH domain of said anti-CD3 antibody is linked to a CH1 orCL domain of said anti-BCMA antibody. Preferably a VL domain of saidanti-CD3 antibody is linked to a CH1 or CL domain of said anti-BCMAantibody.

Preferably the bispecific antibody comprises not more than one Fabfragment of an anti-CD3 antibody, not more than two Fab fragments of ananti-BCMA antibody and not more than one Fc part, preferably a human Fcpart. Preferably not more than one Fab fragment of an anti-CD3 antibodyand not more than one Fab fragment of an anti-BCMA antibody are linkedto the Fc part and linking is performed via C-terminal binding of theFab fragment(s) to the hinge region. Preferably the second Fab fragmentof an anti-BCMA antibody is linked via its C-terminus either to theN-terminus of the Fab fragment of an anti-CD3 antibody or to the hingeregion of the Fc part. The preferred bispecific antibodies are shown inFIG. 3.

Especially preferred are these bispecific antibodies comprising only theFab fragments and the Fc part as specified:

Fab BCMA-Fc-Fab CD3-Fab BCMA (bispecific format 3.1)

Fc-Fab CD3-Fab BCMA (bispecific format 3.2),

Fab CD3-Fab BCMA (bispecific format 3.3),

Fab CD3-Fab BCMA-Fab BCMA (bispecific format 3.4),

Preferably the bispecific antibody comprises a second Fab fragment ofsaid anti-BCMA antibody chemically linked with its C-terminus to theN-terminus of the first Fab fragment of said anti-BCMA antibody.Preferably a VH domain of said first anti-BCMA antibody is linked to aCH1 or CL domain of said second anti-BCMA antibody. Preferably a VLdomain of said first anti-BCMA antibody is linked to a CH1 or CL domainof said second anti-BCMA antibody.

Preferably the bispecific antibody comprises a Fc part linked with itsN-terminus to the C-terminus of said anti-CD3 antibody. Preferably thebispecific antibody comprises a Fc part linked with its first N-terminusto the C-terminus of said anti-CD3 antibody and a second Fab fragment ofsaid anti-BCMA antibody linked with its C-terminus to the secondN-terminus of the Fc part. Preferably the CL domain of the CD3 antibodyFab fragment is linked to the hinge region of the Fc part. Preferablythe CH1 domain of the BCMA antibody Fab fragment is linked to the hingeregion of the Fc part.

The Fab fragments are chemically linked together by the use of anappropriate linker according to the state of the art. Preferably a(Gly₄-Ser₁)₃ linker is used (Desplancq D K et al., Protein Eng. 1994August; 7(8):1027-33 and Mack M. et al., PNAS Jul. 18, 1995 vol. 92 no.15 7021-7025).

The antibodies according to the invention mentioned above are preferablycharacterized by the features mentioned below, especially in regard tothat binding of said antibody is not reduced by 100 ng/ml APRIL andpreferably by BAFF for more than 20% and does not alter APRIL-dependentNF-κB activation for more than 20%, with and without APRIL andpreferably with and without BAFF for more than 20%.

The invention relates to a bispecific antibody specifically binding tothe two targets human CD3ε (further named also as “CD3”) and theextracellular domain of human BCMA (further named also as “BCMA”),characterized in

-   -   a) comprising the light chain and heavy chain of an antibody        specifically binding to one of said targets; and    -   b) comprising the light chain and heavy chain of an antibody        specifically binding to the other one of said targets, wherein        the variable domains VL and VH or the constant domains CL and        CH1 are replaced by each other, and characterized in that    -   c) the binding of said antibody specifically binding to human        BCMA is not reduced by 100 ng/ml APRIL for more than 20%        measured in an ELISA assay as OD at 405 nm compared to the        binding of said antibody to human BCMA without APRIL,    -   d) said antibody does not alter APRIL-dependent NF-κB activation        for more than 20%, as compared to APRIL alone, and    -   e) said antibody does not alter NF-κB activation without APRIL        for more than 20%, as compared without said antibody.

The naked antibody specifically binding to BCMA from which the anti-BCMAantibody portion of the bispecific antibody according to the inventionis derived also shows the above mentioned features c) to e).

Preferably the antibody is further characterized in that the binding ofsaid antibody to human BCMA is not reduced by 100 ng/ml APRIL for morethan 15%, measured in said ELISA. Preferably the antibody is furthercharacterized in that the binding of said antibody to human BCMA is notreduced by 1000 ng/ml APRIL and not reduced by 1000 ng/ml, for more than20%, measured in said ELISA. Preferably the antibody is furthercharacterized in that the binding of said antibody to human BCMA is notreduced by 1000 ng/ml APRIL for more than 15%, measured in said ELISA.

Preferably the antibody according to the invention does not alterAPRIL-dependent NF-κB activation for more than 15%. Preferably theantibody according to the invention does not alter NF-κB activationwithout APRIL for more than 15%.

The invention relates to a bispecific antibody specifically binding tothe two targets human CD3ε (further named also as “CD3”) and theextracellular domain of human BCMA (further named also as “BCMA”),characterized in

-   -   a) comprising the light chain and heavy chain of an antibody        specifically binding to one of said targets; and    -   b) comprising the light chain and heavy chain of an antibody        specifically binding to the other one of said targets, wherein        the variable domains VL and VH or the constant domains CL and        CH1 are replaced by each other, and characterized in that    -   c) the binding of said antibody specifically binding to human        BCMA is not reduced by 100 ng/ml APRIL and not reduced by 100        ng/ml BAFF for more than 20% measured in an ELISA assay as OD at        405 nm compared to the binding of said antibody to human BCMA        without APRIL or BAFF respectively,    -   d) said antibody does not alter APRIL-dependent NF-κB activation        for more than 20%, as compared to APRIL alone,    -   e) said antibody does not alter BAFF-dependent NF-κB activation        for more than 20%, as compared to BAFF alone, and    -   f) said antibody does not alter NF-κB activation without BAFF        and APRIL for more than 20%, as compared without said antibody.

The naked antibody specifically binding to BCMA from which the anti-BCMAantibody portion of the bispecific antibody according to the inventionis derived also shows the above mentioned features c) to f).

Preferably the antibody portion specifically binding to human CD3 ischaracterized in comprising a variable domain VH comprising the heavychain CDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1,CDR2 and CDR3 and a variable domain VL comprising the light chain CDRsof SEQ ID NO: 4, 5 and 6 as respectively light chain CDR1, CDR2 and CDR3of the anti CD3ε antibody (CDR MAB CD3). Preferably the antibody portionspecifically binding to human CD3 is characterized in that the variabledomains are of SEQ ID NO:7 and 8 (VHVL MAB CD3).

Preferably the antibody portion, preferably the Fab fragment,specifically binding to human BCMA is characterized in comprising avariable domain VH comprising the heavy chain CDRs of SEQ ID NO: 37 to45, 47 to 55 and 57 to 65 as respectively heavy chain CDR1, CDR2 andCDR3 and a variable domain VL comprising the light chain CDRs of SEQ IDNO: 67 to 75, 77 to 85, and 87 to 95 as respectively light chain CDR1,CDR2 and CDR3 of the anti BCMA antibody. Preferably the antibody portionspecifically binding to human BCMA is characterized in that the variabledomain VH is selected from the group of SEQ ID NO:17 to 25 and thevariable domain VL is selected from the group of SEQ ID NO:27 to 35respectively.

Preferably the antibody portion, preferably the Fab fragment,specifically binding to human BCMA is characterized in comprising aCDR1H of SEQ ID NO:37, a CDR2H of SEQ ID NO:47, a CDR3H of SEQ ID NO: 57and a CDR1L of SEQ ID NO:67, a CDR2L of SEQ ID NO:77, a CDR3L of SEQ IDNO: 87 (CDR MAB 13C2). Preferably the antibody portion, preferably theFab fragment, specifically binding to human BCMA is characterized incomprising a CDR1H of SEQ ID NO:38, a CDR2H of SEQ ID NO:48, a CDR3H ofSEQ ID NO: 58 and a CDR1L of SEQ ID NO:68, a CDR2L of SEQ ID NO:78, aCDR3L of SEQ ID NO: 88 (CDR MAB 17A5). Preferably the antibody portion,preferably the Fab fragment, specifically binding to human BCMA ischaracterized in comprising a CDR1H of SEQ ID NO:39, a CDR2H of SEQ IDNO:49, a CDR3H of SEQ ID NO: 59 and a CDR1L of SEQ ID NO:69, a CDR2L ofSEQ ID NO:79, a CDR3L of SEQ ID NO: 89 (CDR MAB 83A10). Preferably theantibody portion, preferably the Fab fragment, specifically binding tohuman BCMA is characterized in comprising a CDR1H of SEQ ID NO:40, aCDR2H of SEQ ID NO:50, a CDR3H of SEQ ID NO: 60 and a CDR1L of SEQ IDNO:70, a CDR2L of SEQ ID NO:80, a CDR3L of SEQ ID NO: 90 (CDR MAB 13A4).Preferably the antibody portion, preferably the Fab fragment,specifically binding to human BCMA is characterized in comprising aCDR1H of SEQ ID NO:41, a CDR2H of SEQ ID NO:51, a CDR3H of SEQ ID NO: 61and a CDR1L of SEQ ID NO:71, a CDR2L of SEQ ID NO:81, a CDR3L of SEQ IDNO: 91 (CDR MAB 13D2). Preferably the antibody portion, preferably theFab fragment, specifically binding to human BCMA is characterized incomprising a CDR1H of SEQ ID NO:42, a CDR2H of SEQ ID NO:52, a CDR3H ofSEQ ID NO: 62 and a CDR1L of SEQ ID NO:72, a CDR2L of SEQ ID NO:82, aCDR3L of SEQ ID NO: 92 (CDR MAB 14B11). Preferably the antibody portion,preferably the Fab fragment, specifically binding to human BCMA ischaracterized in comprising a CDR1H of SEQ ID NO:43, a CDR2H of SEQ IDNO:53, a CDR3H of SEQ ID NO: 63 and a CDR1L of SEQ ID NO:73, a CDR2L ofSEQ ID NO:83, a CDR3L of SEQ ID NO: 93 (CDR MAB 14E1). Preferably theantibody portion, preferably the Fab fragment, specifically binding tohuman BCMA is characterized in comprising a CDR1H of SEQ ID NO:44, aCDR2H of SEQ ID NO:54, a CDR3H of SEQ ID NO: 64 and a CDR1L of SEQ IDNO:74, a CDR2L of SEQ ID NO:84, a CDR3L of SEQ ID NO: 94 (CDR MAB29B11).Preferably the antibody portion, preferably the Fab fragment,specifically binding to human BCMA is characterized in comprising aCDR1H of SEQ ID NO:45, a CDR2H of SEQ ID NO:55, a CDR3H of SEQ ID NO: 65and a CDR1L of SEQ ID NO:75, a CDR2L of SEQ ID NO:85, a CDR3L of SEQ IDNO: 95 (CDR MAB29F3).

A further embodiment of the invention is characterized in that thebispecific antibody specifically binding to CD3ε and BCMA consists ofCDR MAB CD3 and a CDR MAB, selected from the group consisting of 13C2,17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11 and 29F3 of the bispecificformat 3.1. A further embodiment of the invention is characterized inthat the bispecific antibody specifically binding to CD3ε and BCMA″)consists of CDR MAB CD3 and a CDR MAB, selected from the groupconsisting of 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11 and 29F3of the bispecific format 3.2. A further embodiment of the invention ischaracterized in that the bispecific antibody specifically binding toCD3ε and BCMA″) consists of CDR MAB CD3 and a CDR MAB, selected from thegroup consisting of 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11and 29F3 of the bispecific format 3.3. A further embodiment of theinvention is characterized in that the bispecific antibody specificallybinding to CD3ε and BCMA″) consists of CDR MAB CD3 and a CDR MAB,selected from the group consisting of 13C2, 17A5, 83A10, 13A4, 13D2,14B11, 14E1, 29B11 and 29F3 of the bispecific format 3.4.

Preferably the antibody portion, preferably the Fab fragment,specifically binding to human BCMA is characterized in comprising a VHselected from the group consisting of SEQ ID NO: 17 to 25 and/or incomprising a VL selected from the group consisting of SEQ ID NO: 27 to35.

Preferably the antibody portion, preferably the Fab fragment,specifically binding to human BCMA is characterized in comprising a VHof SEQ ID NO: 17 and a VL of SEQ ID NO: 27 (VHVL MAB 13C2). Preferablythe antibody portion, preferably the Fab fragment, specifically bindingto human BCMA is characterized in comprising a VH of SEQ ID NO: 18 and aVL of SEQ ID NO: 28 (VHVL MAB 17A5). Preferably the antibody portion,preferably the Fab fragment, specifically binding to human BCMA ischaracterized in comprising a VH of SEQ ID NO: 19 and a VL of SEQ ID NO:29 (VHVL MAB 83A10). Preferably the antibody portion, preferably the Fabfragment, specifically binding to human BCMA is characterized incomprising a VH of SEQ ID NO: 20 and a VL of SEQ ID NO: 30 (VHVL MAB13A4). Preferably the antibody portion, preferably the Fab fragment,specifically binding to human BCMA is characterized in comprising a VHof SEQ ID NO: 21 and a VL of SEQ ID NO: 31 (VHVL MAB13D2). Preferablythe antibody portion, preferably the Fab fragment, specifically bindingto human BCMA is characterized in comprising a VH of SEQ ID NO: 22 and aVL of SEQ ID NO: 32 (VHVL MAB 14B11). Preferably the antibody portion,preferably the Fab fragment, specifically binding to human BCMA ischaracterized in comprising a VH of SEQ ID NO: 23 and a VL of SEQ ID NO:33 (VHVL MAB 14E1). Preferably the antibody portion, preferably the Fabfragment, specifically binding to human BCMA is characterized incomprising a VH of SEQ ID NO: 24 and a VL of SEQ ID NO: 34 (VHVLMAB29B11). Preferably the antibody portion, preferably the Fab fragment,specifically binding to human BCMA is characterized in comprising a VHof SEQ ID NO: 25 and a VL of SEQ ID NO: 35 (VHVL MAB 29F3).

In a further embodiment of the invention, the antibody portion,preferably the Fab fragment, specifically binding to human BCMA ischaracterized in comprising a CDR1H of SEQ ID NO:46, a CDR2H of SEQ IDNO:56, a CDR3H of SEQ ID NO: 66 and a CDR1L of SEQ ID NO:76, a CDR2L ofSEQ ID NO:86, a CDR3L of SEQ ID NO: 96 (CDR MAB 13A7). In a furtherembodiment of the invention the antibody portion, preferably the Fabfragment, specifically binding to human BCMA is characterized incomprising a VH of SEQ ID NO: 26 and a VL of SEQ ID NO: 36 (VHVLMAB13A7). The binding of antibody MAB 13A7 is reduced by 100 ng/ml APRILfor more than 20% measured in an ELISA assay.

A further embodiment of the invention is characterized in that thebispecific antibody specifically binding to CD3ε and BCMA consists ofVHVL MAB CD3 and a VHVL MAB, selected from the group consisting of 13C2,17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, and 29F3 of the bispecificformat 3.1. A further embodiment of the invention is characterized inthat the bispecific antibody specifically binding to CD3ε and BCMA″)consists of VHVL MAB CD3 and a VHVL MAB, selected from the groupconsisting of 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, and29F3 of the bispecific format 3.2. A further embodiment of the inventionis characterized in that the bispecific antibody specifically binding toCD3ε and BCMA″) consists of VHVL MAB CD3 and a VHVL MAB, selected fromthe group consisting of 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1,29B11, 29F3, and 13A7 of the bispecific format 3.3. A further embodimentof the invention is characterized in that the bispecific antibodyspecifically binding to CD3ε and BCMA″) consists of VHVL MAB CD3 and aVHVL MAB, selected from the group consisting of 13C2, 17A5, 83A10, 13A4,13D2, 14B11, 14E1, 29B11, and 29F3 of the bispecific format 3.4.

Preferably the bispecific antibody according to the invention ischaracterized in that the CH3 domain of one heavy chain and the CH3domain of the other heavy chain each meet at an interface whichcomprises an original interface between the antibody CH3 domains;wherein said interface is altered to promote the formation of thebispecific antibody, wherein the alteration is characterized in that:

a) the CH3 domain of one heavy chain is altered, so that within theoriginal interface the CH3 domain of one heavy chain that meets theoriginal interface of the CH3 domain of the other heavy chain within thebispecific antibody, an amino acid residue is replaced with an aminoacid residue having a larger side chain volume, thereby generating aprotuberance within the interface of the CH3 domain of one heavy chainwhich is positionable in a cavity within the interface of the CH3 domainof the other heavy chain and

b) the CH3 domain of the other heavy chain is altered, so that withinthe original interface of the second CH3 domain that meets the originalinterface of the first CH3 domain within the bispecific antibody anamino acid residue is replaced with an amino acid residue having asmaller side chain volume, thereby generating a cavity within theinterface of the second CH3 domain within which a protuberance withinthe interface of the first CH3 domain is positionable.

Preferably the antibody according to the invention is characterized inthat said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), tryptophan (W). Preferably the antibody according to theinvention is characterized in that said amino acid residue having asmaller side chain volume is selected from the group consisting ofalanine (A), serine (S), threonine (T), valine (V). Preferably theantibody according to the invention is characterized in that both CH3domains are further altered by the introduction of cysteine (C) as aminoacid in the corresponding positions of each CH3 domain. Preferably theantibody according to the invention is characterized in that one of theconstant heavy chain domains CH3 of both heavy chains is replaced by aconstant heavy chain domain CH1; and the other constant heavy chaindomain CH3 is replaced by a constant light chain domain CL.

Preferably the antibody is further characterized in that the binding ofsaid antibody to human BCMA is not reduced by 100 ng/ml APRIL and notreduced by 100 ng/ml BAFF for more than 15%, measured in said ELISA.Preferably the antibody is further characterized in that the binding ofsaid antibody to human BCMA is not reduced by 1000 ng/ml APRIL and notreduced by 1000 ng/ml, for more than 20%, measured in said ELISA.Preferably the antibody is further characterized in that the binding ofsaid antibody to human BCMA is not reduced by 1000 ng/ml, APRIL and notreduced by 1000 ng/ml BAFF for more than 15%, measured in said ELISA.

Preferably the antibody according to the invention does not alterAPRIL-dependent NF-κB activation for more than 15%. Preferably theantibody according to the invention does not alter BAFF-dependent NF-κBactivation for more than 15%. Preferably the antibody according to theinvention does not alter NF-κB activation without APRIL and BAFF formore than 15%.

Preferably the antibody according to the invention is characterized inthat its binding to BCMA is not reduced by APRIL and preferably notreduced by BAFF for more than 25%, preferably not more than 20%,preferably not more than 10%, measured as binding of said antibody in aconcentration of 140 nM, preferably 50 nM, and preferably 5 nM toNCI-H929 cells (ATCC® CRL9068™) in presence or absence of APRIL orrespectively BAFF in a concentration of 2.5 μg/ml compared to thebinding of said antibody to NCI-H929 cells without APRIL or BAFFrespectively.

Preferably the antibody according to the invention is furthercharacterized in that it binds also specifically to cynomolgus BCMA.

A further embodiment of the invention is a method for the preparation ofa bispecific antibody according to the invention comprising the steps of

-   -   a) transforming a host cell with vectors comprising nucleic acid        molecules encoding the light chain and heavy chain of an        antibody specifically binding to the first target and vectors        comprising nucleic acid molecules encoding the light chain and        heavy chain of an antibody specifically binding to the second        target, wherein the variable domains VL and VH or the constant        domains CL and CH1 are replaced by each other;    -   b) culturing the host cell under conditions that allow synthesis        of said antibody molecule; and    -   c) recovering said antibody molecule from said culture.

A further embodiment of the invention is a host cell comprising vectorscomprising nucleic acid molecules encoding the light chain and heavychain of an antibody specifically binding to the first target andvectors comprising nucleic acid molecules encoding the light chain andheavy chain of an antibody specifically binding to the second target,wherein the variable domains VL and VH or the constant domains CL andCH1 are replaced by each other.

A further preferred embodiment of the invention is a pharmaceuticalcomposition comprising an antibody according to the invention and apharmaceutically acceptable excipient.

A further embodiment of the invention is a diagnostic compositioncomprising an antibody according to the invention.

A further preferred embodiment of the invention is a pharmaceuticalcomposition comprising an antibody according to the invention for use asa medicament. A further preferred embodiment of the invention is apharmaceutical composition comprising an antibody according to theinvention for use as a medicament in the treatment of plasma celldisorders. A further preferred embodiment of the invention is apharmaceutical composition comprising an antibody according to theinvention for use as a medicament in the treatment of Multiple Myeloma.A further embodiment of the invention is an antibody according to theinvention for the treatment of plasma cell disorders like MultipleMyeloma MM or other B-cell disorders expressing BCMA. MM is a B-cellmalignancy characterized by a monoclonal expansion and accumulation ofabnormal plasma cells in the bone marrow compartment. MM also involvescirculating clonal B cells with same IgG gene rearrangement and somatichypermutation. MM arises from an asymptomatic, premalignant conditioncalled monoclonal gammopathy of unknown significance (MGUS),characterized by low levels of bone marrow plasma cells and a monoclonalprotein. MM cells proliferate at low rate. MM results from a progressiveoccurrence of multiple structural chromosomal changes (e.g. unbalancedtranslocations). MM involves the mutual interaction of malignant plasmacells and bone marrow microenvironment (e.g. normal bone marrow stromalcells). Clinical signs of active MM include monoclonal antibody spike,plasma cells overcrowding the bone marrow, lytic bone lesions and bonedestruction resulting from overstimulation of osteoclasts (Dimopulos &Terpos, Ann Oncol 2010; 21 suppl 7: vii143-150). Another B-cell disorderinvolving plasma cells i.e. expressing BCMA is systemic lupuserythematosus (SLE), also known as lupus. SLE is a systemic, autoimmunedisease that can affect any part of the body and is represented with theimmune system attacking the body's own cells and tissue, resulting inchronic inflammation and tissue damage. It is a Type IIIhypersensitivity reaction in which antibody-immune complexes precipitateand cause a further immune response (Inaki & Lee, Nat Rev Rheumatol2010; 6: 326-337). A further preferred embodiment of the invention ispharmaceutical composition comprising an antibody according to theinvention for use as a medicament in the treatment of systemic lupuserythematosus.

In regard to bispecific antibodies against BCMA and CD3 the inventorsrecognize that a bispecific antibody against BCMA and capable of bindingspecifically to an activating T cell antigen (BCMA-TCB) which 1) is inan ELISA assay not reduced more than 20% in its binding to BCMA by APRILconcentrations relevant in patients with Multiple Myeloma, 2) preferablyis in an ELISA assay not reduced by more than 20% in its binding to BCMAby BAFF concentrations relevant in patients with Multiple Myeloma,avoids that the efficacy of the BCMA-TCB to eradicate BCMA-positivetumor cells in MM patients is negatively affected by the concentrationof APRIL and BAFF in the serum or at the tumor (see also FIGS. 1 and 2and descriptions to FIGS. 1 and 2). In addition the inventors recognizethat a bispecific antibody against BCMA and capable of bindingspecifically to an activating T cell antigen (BCMA-TCB) which 1) is in aH929 cell based flow cytometry assay not reduced more than 25% in itsbinding to BCMA by APRIL concentrations which can be relevant inpatients with Multiple Myeloma, 2) preferably is in a H929 cell basedflow cytometry assay not reduced more than 25% in its binding to BCMA byAPRIL concentrations which can be relevant in patients with MultipleMyeloma, avoids that the efficacy of the BCMA-TCB to eradicateBCMA-positive tumor cells in MM patients is negatively affected by theconcentration of APRIL and BAFF in the serum or at the tumor (see alsoFIGS. 1 and 2 and descriptions to FIGS. 1 and 2).

In addition, the inventors recognize that a bispecific antibody againstBCMA and capable of binding specifically to an activating T cell antigen(BCMA-TCB) which 1) does not block or increase APRIL-dependent NF-κBactivation, 2) preferably does not block or increase BAFF-dependentNF-κB activation, and 3) does not induce NF-κB activation without APRILand preferably without BAFF avoids that the efficacy of the BCMA-TCB toeradicate BCMA-positive tumor cells in MM patients is negativelyaffected by the concentration of APRIL and BAFF in the serum or at thetumor. In addition as the BCMA-TCB does not induce NF-κB activationwithout APRIL and preferably without BAFF, activation and increase ofsurvival of BCMA-positive resp. tumor cells does not occur in the casethat the BCMA-TCB for whatever reasons does not kill the tumor cells,e.g. by not binding to CD3 but only to tumor cells. In addition receptorinternalization may also not occur which could reduce the efficacy ofBCMA-TCB.

Because efficacy of antibodies usually increases with tumoroccupancy/concentration of TCB, the result with a BCMA-TCB without aBCMA antibody according to this invention could be of considerableinter-patient variability in efficacy (e.g. overall less efficacy, seealso FIGS. 1 and 2). In addition, in patients with high levels of serumAPRIL and BAFF (e.g. multiple myeloma patients) it may not be requiredto increase the dose for an antibody according to this invention as itmay not be affected by ligand competition. In contrast, the doses forother ligand-blocking/competing anti-BCMA antibodies may need to beincreased in those patients.

Another advantage of the antibody according to the invention is anelimination half-life of about 1 to 12 days which allows at least onceor twice/week administration. Preferably the antibody according to theinvention is administered once or twice a week preferably viasubcutaneous administration (e.g. preferably in the dose range of 0.25to 2.5, preferably to 25 mg/m²/week). Due to superior cytotoxicityactivities of the antibody according to the invention it can beadministered at least at the same magnitude of clinical dose range (oreven lower) as compared to conventional monospecific antibodies orconventional bispecific antibodies that are not T cell bispecifics (i.e.do not bind to CD3 on one arm). It is envisaged that for an antibodyaccording to the invention subcutaneous administration is preferred inthe clinical settings (e.g. in the dose range of 1-100 mg/m²/week).

According to the invention OD can be measured at 405 nm or 450 nm(preferably with the same relative results, comparison without APRIL orBAFF). According to the invention OD can be measured with human ormurine APIL or BAFF 450 nm (preferably with the same relative results,comparison without APRIL or BAFF).

DESCRIPTION OF THE FIGURES

FIG. 1. Superior binding properties of a non-ligandblocking/non-competing anti-BCMA antibody vs. aligand-blocking/competing anti-BCMA antibody; or a non-ligandblocking/non-competing anti-BCMA containing TCB vs. aligand-blocking/competing anti-BCMA containing TCB on plate-bound-BCMAcells by ELISA. In this graph, increasing concentrations (i.e. 10, 100,1000 ng/mL) of soluble APRIL or BAFF representative of the levels foundin the blood and bone marrow of multiple myeloma patients does not alterthe binding of a non-ligand blocking/non-competing anti-BCMA antibody ornon-ligand blocking/non-competing anti-BCMA containing TCB toplate-bound-BCMA (continuous line). In contrast, the high concentrations(i.e. 100 ng/mL to 1000 ng/mL) of soluble APRIL or BAFF representativeof the levels found in the blood and bone marrow of multiple myelomapatients reduce the binding of a ligand blocking/competing anti-BCMAantibody or ligand blocking/competing anti-BCMA containing TCB toplate-bound-BCMA (dotted line). The concentration of anti-BCMAantibodies or anti-BCMA containing TCB with different properties ispreferably concentration(s) ranging from 0.1 pM to 200 nM as the levelsof add-on circulating APRIL or BAFF range from 1 ng/mL (healthy normal)to 100 ng/mL (MM, blood) and beyond (MM, tumor in bone marrow).

FIG. 2. Superior potency in redirected T cell cytotoxicity ofBCMA-expressing MM cells mediated by a T cell bispecific antibodycontaining a non-ligand blocking/non-competing anti-BCMA antibody vs. aligand-blocking/competing anti-BCMA antibody in a LDH release assay. Inthis graph, increasing concentrations (i.e. 10, 100, 1000 ng/mL) ofsoluble APRIL or BAFF representative of the levels found in the bloodand bone marrow of multiple myeloma patients does not alter the killingpotency of a T cell bispecific antibody containing a non-ligandblocking/non-competing anti-BCMA antibody specific to BCMA-expressing MMcells (continuous line). In contrast, the high concentrations (i.e. 100ng/mL to 1000 ng/mL) of soluble APRIL or BAFF representative of thelevels found in the blood and bone marrow of multiple myeloma patientsdecrease the killing potency of a T cell bispecific antibody containinga ligand blocking/competing anti-BCMA antibody specific toBCMA-expressing MM cells (dotted line). The concentration of T cellbispecifics with anti-BCMA antibody with different properties ispreferably concentration(s) ranging from 0.1 pM to 200 nM as the levelsof add-on circulating APRIL or BAFF range from 1 ng/mL (healthy normal)to 100 ng/mL (MM, blood) and beyond (MM, tumor in bone marrow).

FIG. 3. Preferred bispecific antibodies comprising only the Fabfragments (specific to CD3 and BCMA) and the Fc part as specified: (1)Fab BCMA-Fc-Fab CD3-Fab BCMA; (2) Fc-Fab CD3-Fab BCMA; (3) Fab CD3-FabBCMA; (4) Fab CD3-Fab BCMA-Fab BCMA. Preferably, the Fabs CD3 include aCH1-CL crossover to reduce LC mispairing and side-products. Fab CD3 andFab BCMA are linked to each other with flexible linkers.

FIG. 4. BCMA expression on multiple myeloma cell lines. Increase ofmedian fluorescence intensity upon binding of increasing concentrationsof the anti-BCMA antibody (from 0.3 to 10 μg/mL) to H929 cells asdetected by flow cytometry.

FIG. 5. Binding of anti-BCMA antibodies on BCMA-positive multiplemyeloma cells. Mean fluorescence intensity for anti-BCMA IgG clonesplotted in function of anti-BCMA antibody concentrations (from 0.2 to 40μg/mL); (A) clones 13C2, 17A5, 83A10 on H929 cells, (B) clones 13C2,17A5, 83A10 on MKN45 cells, (C) clones 13A4, 13D2, 14E1, 13A7, 14B11 onH929 cells (D) clones 13A4, 13D2, 14E1, 13A7, 14B11 on MKN45 cells.

FIG. 6. Competition ELISA. ELISA results of 7 selected anti-BCMA Fabclones (13C2, 17A5, 83A19, 13A4, 13D2, 29B11, 13A7), at saturatingconcentrations of 500 or 1000 nM, binding to immobilized human BCMA inthe presence of a concentration range of murine APRIL (from 1.56 to 100nM) are shown. In case of non-competition, signals remain constantwithin the variability of the assay across the concentration range andsignals in the presence of murine APRIL are comparable to those from thecontrol wells where no murine APRIL was added. In case of competition aconcentration dependent reduction of the signal is measured.

FIG. 7. Binding competition by FACS. Competition of Δ-APRIL withanti-BCMA antibodies detected by flow cytometry. Relative medianfluorescence intensity of Δ-APRIL (FITC signal) used at a concentrationof 1000 ng/mL detected in function of concentrations (1, 16, and 40μg/mL) of anti-BCMA antibody clones 13A4, 13D2, 14E1, 14B11 on H929cells. The median fluorescence intensity upon binding of Δ-APRIL inpresence of the isotype control was set to one; the other signals werenormalized to it. The detection of APRIL binding to BCMA-positive H929cells in the presence of anti-BCMA antibodies was measured via anti-HAfluorochrome-conjugated antibody.

FIG. 8. Binding competition by FACS. Competition of anti-BCMA antibodieswith Δ-APRIL detected by flow cytometry. The relative medianfluorescence intensity of anti-BCMA antibody (Alexa.Fluor 647 signal)used at a concentration of 40 μg/mL for anti-BCMA antibody clones 13A4,13C7, 13D2, 14B11, 17A5, 83A10 on RPMI cells detected in absence orpresence of Δ-APRIL 1000 ng/mL. The median fluorescence intensity uponbinding of anti-BCMA antibodies in absence of Δ-APRIL was set to one;the other signals respective to the anti-BCMA antibody in presence ofΔ-APRIL were normalized to it. The detection of anti-BCMA antibodiesbinding to BCMA-positive RPMI cells in the presence of Δ-APRIL wasmeasured via anti-human Fc fluorochrome-conjugated antibody.

FIG. 9. Competition of anti-BCMA antibodies with Δ-APRIL aftersimultaneous incubation detected by flow cytometry. (A) The meanfluorescence intensity and the relative fluorescence signal (Alexa.Fluor647 signal) of the anti-BCMA antibody clones 14B11, 13D2, 13A4, 17A5 and83A10 at the concentration of 20 μg/mL in presence or absence of 2.5μg/mL Δ-APRIL or (B) the mean fluorescence intensity and the relativefluorescence signal of Δ-APRIL (FITC signal) at a concentration of 2.5μg/mL Δ-APRIL and the anti-BCMA antibody clone 83A10 (20 μg/mL)(Alexa.Fluor 647 signal) were measured. Detection of anti-BCMA antibodyin presence of Δ-APRIL with FITC conjugated anti-human Fc antibody wasnormalized to the signal of anti-BCMA antibody clone in absence Δ-APRIL.Detection of Δ-APRIL in presence of the anti-BCMA antibody clone withAlexa.Fluor 647 conjugated anti-HA antibody was normalized to Δ-APRILsignal in presence of the isotype control.

DETAILED DESCRIPTION OF THE INVENTION

The term “BCMA” as used herein relates to human B cell maturationtarget, also known as BCMA; TR17_HUMAN, TNFRSF17 (UniProt Q02223), whichis a member of the tumor necrosis receptor superfamily that ispreferentially expressed in differentiated plasma cells. Theextracellular domain of BCMA consists according to UniProt of aminoacids 1-54 (or 5-51). The term “antibody against BCMA, anti BCMAantibody” as used herein relates to an antibody specifically binding toBCMA.

The term “CD3ε or CD3” as used herein relates to human CD3ε describedunder UniProt P07766 (CD3E_HUMAN). The term “antibody against CD3, antiCD3 antibody” relates to an antibody binding to CD3ε. Preferably theantibody comprises a variable domain VH comprising the heavy chain CDRsof SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1, CDR2 and CDR3and a variable domain VL comprising the light chain CDRs of SEQ ID NO:4, 5 and 6 as respectively light chain CDR1, CDR2 and CDR3. Preferablythe antibody comprises the variable domains of SEQ ID NO:7 (VH) and SEQID NO:8 (VL). The term “antibody against CD3, anti CD3 antibody” as usedherein relates to an antibody specifically binding to CD3.

“Specifically binding to CD3 or BCMA” refer to an antibody that iscapable of binding CD3 or BCMA (the targets) with sufficient affinitysuch that the antibody is useful as a therapeutic agent in targeting CD3or BCMA. In some embodiments, the extent of binding of an anti-CD3 orBCMA antibody to an unrelated, non-CD3 or non-BCMA protein is about10-fold preferably >100-fold less than the binding of the antibody toCD3 or BCMA as measured, e.g., by surface plasmon resonance (SPR) e.g.Biacore®, enzyme-linked immunosorbent (ELISA) or flow cytometry (FACS).Preferably the antibody that binds to CD3 or BCMA has a dissociationconstant (Kd) of 10⁻⁸ M or less, preferably from 10⁻⁸ M to 10⁻¹³ M,preferably from 10⁻⁹ M to 10⁻¹³ M. Preferably the anti-CD3 and/oranti-BCMA antibody binds to an epitope of CD3 and/or BCMA that isconserved among CD3 and/or BCMA from different species, preferably amonghuman and cynomolgus. “Bispecific antibody specifically binding to CD3and BCMA” or “antibody according to the invention” refers to arespective definition for binding to both targets. An antibodyspecifically binding to BCMA (or BCMA and CD3) does not bind to otherhuman antigens. Therefore in an ELISA, OD values for such unrelatedtargets will be equal or lower to that of the limit of detection of thespecific assay, preferably >0.3 ng/mL, or equal or lower to OD values ofcontrol samples without plate-bound-BCMA or with untransfected HEK293cells.

The term “APRIL” as used herein relates to recombinant, truncated murineAPRIL (amino acids 106-241; NP_076006). APRIL can be produced asdescribed in Ryan, 2007 (Mol Cancer Ther; 6 (11): 3009-18).

The term “BAFF” as used herein relates to recombinant, truncated humanBAFF (UniProt Q9Y275 (TN13B_HUMAN) which can be produced as described inGordon, 2003 (Biochemistry; 42 (20): 5977-5983). Preferably a His-taggedBAFF is used according to the invention. Preferably the His-tagged BAFFis produced by cloning a DNA fragment encoding BAFF residues 82-285 intoan expression vector, creating a fusion with an N-terminal His-tagfollowed by a thrombin cleavage site, expressing said vector andcleaving the recovered protein with thrombin.

Anti-BCMA antibodies are analyzed by ELISA for binding to human BCMAusing plate-bound BCMA in the presence and absence of APRIL and/or BAFF.For this assay, an amount of plate-bound BCMA preferably 1.5 μg/mL andconcentration(s) preferably ranging from 1 pM to 200 nM of anti-BCMAantibody are used. A BCMA antibody for which its BCMA binding is notinhibited according to the invention is an anti-BCMA antibody “notinhibiting the binding of APRIL and/or BAFF to human BCMA in an ELISAassay”.

The term “NF-κB” as used herein relates to recombinant NF-κB p50(accession number (P19838).

NF-κB activity is measured by a DNA-binding ELISA of an extract ofNCI-H929 MM cells (CRL-9068™). NCI-H929 MM cells, untreated or treatedwith 0.1 μg/mL TNF-α, 100 ng/mL heat-treated HT-truncated-BAFF, 100ng/mL truncated-BAFF, 0.1 pM to 200 nM isotype control, and with orwithout 0.1 pM to 200 nM of anti-BCMA antibodies are incubated for 20min NF-κB activity is assayed using a functional ELISA that detectschemiluminescent signal from p65 bound to the NF-κB consensus sequence(U.S. Pat. No. 6,150,090).

An antibody that does not block APRIL-dependent NF-κB activation formore than 20% and does not reduce APRIL-dependent NF-κB activation formore than 20% and does not increase APRIL-dependent NF-κB activation formore than 20% is considered “not to alter APRIL-dependent NF-κBactivation” for more than 20% as compared to APRIL-induced NF-κBactivation without an antibody according to the invention (controlgroup); 20% representing the mean standard variability betweenexperiments. Preferably an antibody according to the invention does notalter APRIL-dependent NF-κB activation for more than 15%.

An antibody that does not block BAFF-dependent NF-κB activation for morethan 20% and does not reduce BAFF-dependent NF-κB activation for morethan 20% and does not increase BAFF-dependent NF-κB activation for morethan 20% is considered “not to alter BAFF-dependent NF-κB activation”for more than 20% as compared to BAFF-induced NF-κB activation withoutan antibody according to the invention (control group); 20% representingthe mean standard variability between experiments. Preferably anantibody according to the invention does not alter BAFF-dependent NF-κBactivation for more than 15%.

An antibody that does not block NF-κB activation without APRIL and BAFFfor more than 20% and does not reduce NF-κB activation without APRIL andBAFF for more than 20% and does not increase NF-κB activation withoutAPRIL and BAFF for more than 20% is considered “not to alter NF-κBactivation without APRIL and BAFF” for more than 20% as compared toAPRIL-induced NF-κB activation without an antibody according to theinvention (control group); 20% representing the mean standardvariability between experiments. Preferably an antibody according to theinvention does not alter NF-κB activation without APRIL and BAFF formore than 15%.

Also if an antibody according to the invention is used in large excess,preferably up to 500 nM or 1000 nM binding of said antibody is notreduced by 100 ng/ml APRIL and preferably by BAFF for more than 20% anddoes not alter APRIL-dependent NF-κB activation for more than 20%, withand without APRIL and preferably with and without BAFF for more than20%.

The term “target” as used herein means either BCMA or CD3. The term“first target and second target” means either CD3 as first target andBCMA as second target or means BCMA as first target and CD3 as secondtarget.

The term “antibody” as used herein refers to a monoclonal antibody. Anantibody consists of two pairs of a “light chain” (LC) and a “heavychain” (HC) (such light chain (LC)/heavy chain pairs are abbreviatedherein as LC/HC). The light chains and heavy chains of such antibodiesare polypeptides consisting of several domains. Each heavy chaincomprises a heavy chain variable region (abbreviated herein as HCVR orVH) and a heavy chain constant region. The heavy chain constant regioncomprises the heavy chain constant domains CH1, CH2 and CH3 (antibodyclasses IgA, IgD, and IgG) and optionally the heavy chain constantdomain CH4 (antibody classes IgE and IgM). Each light chain comprises alight chain variable domain VL and a light chain constant domain CL. Thevariable domains VH and VL can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The “constant domains” of theheavy chain and of the light chain are not involved directly in bindingof an antibody to a target, but exhibit various effector functions.

The term “antibody” includes e.g. mouse antibodies, human antibodies,chimeric antibodies, humanized antibodies and genetically engineeredantibodies (variant or mutant antibodies) as long as theircharacteristic properties are retained. Especially preferred are humanor humanized antibodies, especially as recombinant human or humanizedantibodies.

The terms “bispecific antibody” and “antibody according to theinvention” as used herein refer to an antibody in which one of the twopairs of heavy chain and light chain (HC/LC) is specifically binding toCD3 and the other one is specifically binding to BCMA.

There are five types of mammalian antibody heavy chains denoted by theGreek letters: α, δ, ε, γ, and μ (Janeway C A, Jr et al (2001).Immunobiology. 5th ed., Garland Publishing). The type of heavy chainpresent defines the class of antibody; these chains are found in IgA,IgD, IgE, IgG, and IgM antibodies, respectively (Rhoades R A, Pflanzer RG (2002). Human Physiology, 4th ed., Thomson Learning). Distinct heavychains differ in size and composition; α and γ contain approximately 450amino acids, while μ and ε have approximately 550 amino acids. Eachheavy chain has two regions, the constant region and the variableregion. The constant region is identical in all antibodies of the sameisotype, but differs in antibodies of different isotype. Heavy chains γ,α and δ have a constant region composed of three constant domains CH1,CH2, and CH3 (in a line), and a hinge region for added flexibility (WoofJ, Burton D Nat Rev Immunol 4 (2004) 89-99); heavy chains μ and ε have aconstant region composed of four constant domains CH1, CH2, CH3, and CH4(Janeway C A, Jr et al (2001). Immunobiology. 5th ed., GarlandPublishing). The variable region of the heavy chain differs inantibodies produced by different B cells, but is the same for allantibodies produced by a single B cell or B cell clone. The variableregion of each heavy chain is approximately 110 amino acids long and iscomposed of a single antibody domain.

In mammals there are only two types of light chain, which are calledlambda (λ) and kappa (κ). A light chain has two successive domains: oneconstant domain CL and one variable domain VL. The approximate length ofa light chain is 211 to 217 amino acids. Preferably the light chain is akappa (κ) light chain, and the constant domain CL is preferably derivedfrom a kappa (K) light chain (the constant domain CK).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The “antibodies” according to the invention can be of any class (e.g.IgA, IgD, IgE, IgG, and IgM, preferably IgG or IgE), or subclass (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1), whereby bothantibodies, from which the bivalent bispecific antibody according to theinvention is derived, have an Fc part of the same subclass (e.g. IgG1,IgG4 and the like, preferably IgG1), preferably of the same allotype(e.g. Caucasian).

A “Fab fragment of an antibody” as used herein is a fragment on anantibody that binds to antigens. It is composed of one constant and onevariable domain of each of the heavy chain (CH1 and VH) and the lightchain (CL and VL). According to the invention the domains of the heavyand light chain of a Fab fragment are not chemically linked together andare therefore no scFvs (single chain variable fragments).

A “Fc part of an antibody” is a term well known to the skilled artisanand defined on the basis of papain cleavage of antibodies. Theantibodies according to the invention contain as Fc part, preferably aFc part derived from human origin and preferably all other parts of thehuman constant regions. The Fc part of an antibody is directly involvedin complement activation, C1q binding, C3 activation and Fc receptorbinding. While the influence of an antibody on the complement system isdependent on certain conditions, binding to C1q is caused by definedbinding sites in the Fc part. Such binding sites are known in the stateof the art and described e.g. by Lukas, T J., et al., J. Immunol. 127(1981) 2555-2560; Brunhouse, R., and Cebra, J. J., MoI. Immunol. 16(1979) 907-917; Burton, D. R., et al., Nature 288 (1980) 338-344;Thommesen, J. E., et al., MoI. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J.Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995)319-324; and EP 0 307 434. Such binding sites are e.g. L234, L235, D270,N297, E318, K320, K322, P331 and P329 (numbering according to EU indexof Kabat, see below). Antibodies of subclass IgG1, IgG2 and IgG3 usuallyshow complement activation, C1q binding and C3 activation, whereas IgG4do not activate the complement system, do not bind C1q and do notactivate C3. Preferably the Fc part is a human Fc part. Preferably theFc part is a human IgG1Fc part.

Preferably the antibody according to the invention comprises as Fc partan Fc variant of a wild-type human IgG Fc region, said Fc variantcomprising an amino acid substitution at position Pro329 and at leastone further amino acid substitution, wherein the residues are numberedaccording to the EU index of Kabat, and wherein said antibody exhibits areduced affinity to the human FcγRIIIA and/or FcγRIIA and for FcγRIcompared to an antibody comprising the wildtype IgG Fc region, andwherein the ADCC induced by said antibody is reduced to at least 20% ofthe ADCC induced by the antibody comprising a wild-type human IgG Fcregion. In a specific embodiment Pro329 of a wild-type human Fc regionin the antibody according to the invention is substituted with glycineor arginine or an amino acid residue large enough to destroy the prolinesandwich within the Fc/Fcγ receptor interface, that is formed betweenthe proline329 of the Fc and tryptophane residues Trp 87 and Tip 110 ofFcγRIII (Sondermann et al.: Nature 406, 267-273 (20 Jul. 2000)). In afurther aspect of the invention the at least one further amino acidsubstitution in the Fc variant is S228P, E233P, L234A, L235A, L235E,N297A, N297D, or P331S and still in another embodiment said at least onefurther amino acid substitution is L234A and L235A of the human IgG1 Fcregion or S228P and L235E of the human IgG4 Fc region. Such Fc variantsare described in detail in WO2012130831.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a murine variable region and a human constant region arepreferred. Other preferred forms of “chimeric antibodies” encompassed bythe present invention are those in which the constant region has beenmodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding. Such chimeric antibodies arealso referred to as “class-switched antibodies”. Chimeric antibodies arethe product of expressed immunoglobulin genes comprising DNA segmentsencoding immunoglobulin variable regions and DNA segments encodingimmunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos.5,202,238 and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the targets notedabove for chimeric antibodies. Other forms of “humanized antibodies”encompassed by the present invention are those in which the constantregion has been additionally modified or changed from that of theoriginal antibody to generate the properties according to the invention,especially in regard to C1q binding and/or Fc receptor (FcR) binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon target challenge (see,e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G., J. MoI. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.MoI. Biol. 222 (1991) 581-597). The techniques of Cole et al. andBoerner et al. are also available for the preparation of humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J.Immunol. 147 (1991) 86-95). As already mentioned for chimeric andhumanized antibodies according to the invention the term “humanantibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation).

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NSO or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The “variable domain” (variable domain of a light chain (VL), variableregion of a heavy chain (VH)) as used herein denotes each of the pair oflight and heavy chains which is involved directly in binding theantibody to the target. The domains of variable human light and heavychains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementarity determiningregions, CDRs). The framework regions adopt a β-sheet conformation andthe CDRs may form loops connecting the β-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe target binding site. The antibody heavy and light chain CDR3 regionsplay a particularly important role in the binding specificity/affinityof the antibodies according to the invention and therefore provide afurther object of the invention.

The terms “hypervariable region” or “target-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for target-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. CDRs on each chain are separated by such framework aminoacids. Especially, CDR3 of the heavy chain is the region whichcontributes most to target binding. CDR and FR regions are determinedaccording to the standard definition of Kabat et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

The constant heavy chain domain CH1 by which the heavy chain domain CH3is replaced can be of any Ig class (e.g. IgA, IgD, IgE, IgG, and IgM),or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The constantlight chain domain CL by which the heavy chain domain CH3 is replacedcan be of the lambda (λ) or kappa (κ) type, preferably the kappa (κ)type.

The term “target” or “target molecule” as used herein are usedinterchangeable and refer to human BCMA and human CD3ε.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of a target that is bound by an antibody.

In general there are two vectors encoding the light chain and heavychain of said antibody specifically binding to the first target, andfurther two vectors encoding the light chain and heavy chain of saidantibody specifically binding to the second target. One of the twovectors is encoding the respective light chain and the other of the twovectors is encoding the respective heavy chain. However in analternative method for the preparation of a bispecific antibodyaccording to the invention, only one first vector encoding the lightchain and heavy chain of the antibody specifically binding to the firsttarget and only one second vector encoding the light chain and heavychain of the antibody specifically binding to the second target can beused for transforming the host cell.

The term “nucleic acid or nucleic acid molecule”, as used herein, isintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham and Van der Eh, Virology 52 (1978) 546ff. However, other methodsfor introducing DNA into cells such as by nuclear injection or byprotoplast fusion may also be used. If prokaryotic cells or cells whichcontain substantial cell wall constructions are used, e.g. one method oftransfection is calcium treatment using calcium chloride as described byCohen S N, et al, PNAS 1972, 69 (8): 2110-2114.

Recombinant production of antibodies using transformation is well-knownin the state of the art and described, for example, in the reviewarticles of Makrides, S. C, Protein Expr. Purif. 17 (1999) 183-202;Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, RJ., MoI. Biotechnol. 16 (2000) 151-161; Werner, R. G., et al.,Arzneimittelforschung 48 (1998) 870-880 as well as in U.S. Pat. No.6,331,415 and U.S. Pat. No. 4,816,567.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

The bispecific antibodies according to the invention are preferablyproduced by recombinant means. Such methods are widely known in thestate of the art and comprise protein expression in prokaryotic andeukaryotic cells with subsequent isolation of the antibody polypeptideand usually purification to a pharmaceutically acceptable purity. Forthe protein expression, nucleic acids encoding light and heavy chains orfragments thereof are inserted into expression vectors by standardmethods. Expression is performed in appropriate prokaryotic oreukaryotic host cells like CHO cells, NSO cells, SP2/0 cells, HEK293cells, COS cells, yeast, or E. coli cells, and the antibody is recoveredfrom the cells (supernatant or cells after lysis). The bispecificantibodies may be present in whole cells, in a cell lysate, or in apartially purified or substantially pure form. Purification is performedin order to eliminate other cellular components or other contaminants,e.g. other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, column chromatography and others wellknown in the art. See Ausubel, F., et al., ed., Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987).

Expression in NSO cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the

DNA sequences being linked are contiguous, and, in the case of asecretory leader, contiguous and in reading frame. However, enhancers donot have to be contiguous. Linking is accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

The bispecific antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA or RNAencoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of the bispecific antibody areprepared by introducing appropriate nucleotide changes into the antibodyDNA, or by nucleotide synthesis. Such modifications can be performed,however, only in a very limited range, e.g. as described above. Forexample, the modifications do not alter the above mentioned antibodycharacteristics such as the IgG isotype and target binding, but mayimprove the yield of the recombinant production, protein stability orfacilitate the purification.

T cell bispecific (TCB) binders have very highconcentration/tumor-cell-receptor-occupancy dependent potency in cellkilling (e.g. EC₅₀ in in vitro cell killing assays in the sub- or lowpicomolar range; Dreier et al. Int J Cancer 2002), T-cell bispecificbinder (TCB) are given at much lower doses than conventionalmonospecific antibodies. For example, blinatumomab (CD19×CD3) is givenat a continuous intravenous dose of 5 to 15 μg/m²/day (i.e. only 0.35 to0.105 mg/m²/week) for treatment of acute lymphocytic leukemia or 60μg/m²/day for treatment of Non Hodgkin Lymphoma, and the serumconcentrations at these doses are in the range of 0.5 to 4 ng/ml(Klinger et al., Blood 2012; Topp et al., J Clin Oncol 2011; Goebeler etal. Ann Oncol 2011). Because low doses of TCB can exert high efficacy inpatients, it is envisaged that for an antibody according to theinvention subcutaneous administration is possible and preferred in theclinical settings (preferably in the dose range of 0.25 to 2.5mg/m²/week). Even at these low concentrations/doses/receptoroccupancies, TCB can cause considerable adverse events (Klinger et al.,Blood 2012). Therefore it is critical to control tumor celloccupancy/coverage. In patients with high and variable levels of serumAPRIL and BAFF (e.g. multiple myeloma patients, Moreaux et al. 2004;Blood 103(8): 3148-3157) number of TCB bound to the tumor cells resp.tumor cell occupancy may be considerably influenced by APRIL/BAFF. Butby using said antibody of this invention, tumor cell occupancyrespectively efficacy/safety it may not be required to increase the dosefor an antibody according to this invention as said antibody may not beaffected by APRIL/BAFF ligand competition. Another advantage of theantibody according to the invention is based on the inclusion of an Fcportion, which increases the elimination half-life to ˜12 days andallows at least once or twice/week administrations as compared to TCBswithout an Fc portion (e.g. blinatumomab) which are required to be givenintravenously and continuously with a pump carried by patients.

TABLE 1 Antibody sequences BCMA SEQ ID NO: antibody VH VL CDR1H CDR2HCDR3H CDR1L CDR2L CDR3L 13C2 17 27 37 47 57 67 77 87 17A5 18 28 38 48 5868 78 88 83A10 19 29 39 49 59 69 79 89 13A4 20 30 40 50 60 70 80 90 13D221 31 41 51 61 71 81 91 14B11 22 32 42 52 62 72 82 92 14E1 23 33 43 5363 73 83 93 29B11 24 34 44 54 64 74 84 94 29F3 25 35 45 55 65 75 85 9513A7 26 36 46 56 66 76 86 96

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered and referred to according toEU numbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63(1969) 78-85; Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md., (1991)).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturers'instructions. General information regarding the nucleotide sequences ofhuman immunoglobulins light and heavy chains is given in: Kabat, E. A.et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed.,NIH Publication No. 91-3242.

Gene Synthesis

Desired gene segments are prepared from oligonucleotides made bychemical synthesis. The 600-1800 bp long gene segments, which areflanked by singular restriction endonuclease cleavage sites, areassembled by annealing and ligation of oligonucleotides including PCRamplification and subsequently cloned via the indicated restrictionsites e.g. Kpnl/Sad or Ascl/Pacl into a pPCRScript (Stratagene) basedpGA4 cloning vector. The DNA sequences of the subcloned gene fragmentsare confirmed by DNA sequencing. Gene synthesis fragments are orderedaccording to given specifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing.

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 is usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

a) For the expression of the described antibodies variants of expressionplasmids for transient expression (e.g. in HEK293 EBNA or HEK293-F)cells based either on a cDNA organization with a CMV-Intron A promoteror on a genomic organization with a CMV promoter are applied. Beside theantibody expression cassette the vectors contain an origin ofreplication which allows replication of this plasmid in E. coli, and—aβ-lactamase gene which confers ampicillin resistance in E. coli. Thetranscription unit of the antibody gene is composed of the followingelements:

-   -   unique restriction site(s) at the 5′ end—the immediate early        enhancer and promoter from the human cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   a immunoglobulin heavy chain signal sequence,    -   the human antibody chain (wildtype or with domain exchange)        either as cDNA or as genomic organization with an the        immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the described antibody chains as describedbelow are generated by PCR and/or gene synthesis and assembled withknown recombinant methods and techniques by connection of the accordingnucleic acid segments e.g. using unique restriction sites in therespective vectors. The subcloned nucleic acid sequences are verified byDNA sequencing. For transient transfections larger quantities of theplasmids are prepared by plasmid preparation from transformed E. colicultures (Nucleobond AX, Macherey-Nagel).

b) Generation of Antibody and Antigen Expression Vectors

The variable region of heavy and light chain DNA sequences weresubcloned in frame with either the human IgG1 constant heavy chain orthe hum IgG1 constant light chain pre-inserted into the respectiverecipient mammalian expression vector. The antibody expression wasdriven by a chimeric MPSV promoter comprising a CMV enhancer and a MPSVpromoter followed by a 5′ UTR, an intron and a kappa MAR element. Thetranscription is terminated by a synthetic polyA signal sequence at the3′ end of the CDS. All vectors carry a 5′-end DNA sequence coding for aleader peptide which targets proteins for secretion in eukaryotic cells.In addition each vector contains an EBV OriP sequence for episomalplasmid replication in EBV EBNA expressing cells.

The antigens that have been used for the phage display selectioncampaigns and to characterize the binding properties of the selectedantibodies were expressed from mammalian antigen expression vectors withpre-inserted DNA sequences coding for C-terminal tags. An Avi tag hasbeen used for in vivo or in vitro biotinylation of the respectiveantigen. For purification and homo- or heterodimerization of the antigena hum IgG1 Fc wt or Fc knob was fused to the C-terminus of the antigenexpression cassette. The antigen expression was driven by a chimericMPSV promoter comprising a CMV enhancer and a MPSV promoter followed bya 5′ UTR, an intron and a kappa MAR element. The transcription wasterminated by a synthetic polyA signal sequence at the 3′ end of theCDS. All vectors carry a 5′-end DNA sequence coding for a leader peptidewhich targets proteins for secretion in eukaryotic cells. In additioneach vector contains an EBV OriP sequence for episomal plasmidreplication in EBV EBNA expressing cells.

Cell Culture Techniques

Standard cell culture techniques are used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Transient Expression in HEK293 Cells

Bispecific antibodies are expressed by transient co-transfection of therespective expression plasmids in adherently growing HEK293-EBNA or inHEK293-F cells growing in suspension as described below.

a) Transient Transfections in HEK293-EBNA System

Bispecific antibodies are expressed by transient co-transfection of therespective expression plasmids (e.g. encoding the heavy and modifiedheavy chain, as well as the corresponding light and modified lightchain) in adherently growing HEK293-EBNA cells (human embryonic kidneycell line 293 expressing Epstein-Barr-Virus nuclear target; Americantype culture collection deposit number ATCC #CRL-10852, Lot. 959 218)cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco)supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco), 2 mML-Glutamine (Gibco), and 250 μg/ml Geneticin (Gibco). For transfectionFuGENE™ 6 Transfection Reagent (Roche Molecular Biochemicals) is used ina ratio of FuGENE™ reagent (μl) to DNA (μg) of 4:1 (ranging from 3:1 to6:1).

Proteins are expressed from the respective plasmids using a molar ratioof (modified and wildtype) light chain and heavy chain encoding plasmidsof 1:1 (equimolar) ranging from 1:2 to 2:1, respectively. Cells arefeeded at day 3 with L-Glutamine ad 4 mM, Glucose [Sigma] and NAA[Gibco]. Bispecific antibody containing cell culture supernatants areharvested from day 5 to 11 after transfection by centrifugation andstored at −200 C. General information regarding the recombinantexpression of human immunoglobulins in e.g. HEK293 cells is given in:Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

b) Transient Transfections in HEK293-F System

Bispecific antibodies are generated by transient transfection of therespective plasmids (e.g. encoding the heavy and modified heavy chain,as well as the corresponding light and modified light chain) using theHEK293-F system (Invitrogen) according to the manufacturer'sinstruction. Briefly, HEK293-F cells (Invitrogen) growing in suspensioneither in a shake flask or in a stirred fermenter in serumfree FreeStyle293 expression medium (Invitrogen) are transfected with a mix of thefour expression plasmids and 293fectin or fectin (Invitrogen). For 2 Lshake flask (Corning) HEK293-F cells are seeded at a density of 1.0×10⁶cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. The day after thecells are transfected at a cell density of ca. 1.5×10⁶ cells/mL with ca.42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmidDNA (1 μg/mL) encoding the heavy or modified heavy chain, respectivelyand the corresponding light chain in an equimolar ratio and B) 20 mlOpti-MEM+1.2 mL 293 fectin or fectin (2 μl/mL). According to the glucoseconsumption glucose solution is added during the course of thefermentation. The supernatant containing the secreted antibody isharvested after 5-10 days and antibodies are either directly purifiedfrom the supernatant or the supernatant is frozen and stored.

Protein Determination

The protein concentration of purified antibodies and derivatives isdetermined by determining the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace et al., Protein Science, 1995, 4, 2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culturesupernatants is estimated by immunoprecipitation with Protein AAgarose-beads (Roche). 60 μL Protein A Agarose beads are washed threetimes in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant is applied to the ProteinA Agarose beads pre-equilibrated in TBS-NP40. After incubation for at 1h at room temperature the beads are washed on an Ultrafree-MC-filtercolumn (Amicon] once with 0.5 mL TBS-NP40, twice with 0.5 mL 2×phosphate buffered saline (2×PBS, Roche) and briefly four times with 0.5mL 100 mM Na-citrate pH 5.0. Bound antibody is eluted by addition of 35μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of the sample iscombined with NuPAGE® Sample Reducing Agent or left unreduced,respectively, and heated for 10 min at 70° C. Consequently, 5-30 μl areapplied to an 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) (with MOPSbuffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidantrunning buffer additive (Invitrogen) for reduced SDS-PAGE) and stainedwith Coomassie Blue.

The concentration of antibodies and derivatives in cell culturesupernatants is quantitatively measured by affinity HPLC chromatography.Briefly, cell culture supernatants containing antibodies and derivativesthat bind to Protein A are applied to an Applied Biosystems Poros A/20column in 200 mM KH₂PO₄, 100 mM sodium citrate, pH 7.4 and eluted fromthe matrix with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an AgilentHPLC 1100 system. The eluted protein is quantified by UV absorbance andintegration of peak areas. A purified standard IgG1 antibody served as astandard.

Alternatively, the concentration of antibodies and derivatives in cellculture supernatants is measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche)are coated with 100 μL/well biotinylated anti-human IgG capture moleculeF(ab′)2<h-Fcγ> BI (Dianova) at 0.1 μg/mL for 1 h at room temperature oralternatively over night at 4° C. and subsequently washed three timeswith 200 μL/well PBS, 0.05% Tween® (PBST, Sigma). 100 μL/well of adilution series in PBS (Sigma) of the respective antibody containingcell culture supernatants is added to the wells and incubated for 1-2 hon a micro titerplate shaker at room temperature. The wells are washedthree times with 200 μL/well PBST and bound antibody is detected with100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as detection antibody for1-2 h on a microtiterplate shaker at room temperature. Unbound detectionantibody is washed away three times with 200 μL/well PBST and the bounddetection antibody is detected by addition of 100 μL ABTS/well.Determination of absorbance is performed on a Tecan Fluor Spectrometerat a measurement wavelength of 405 nm (reference wavelength 492 nm).

Protein Purification

Proteins are purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies are applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies is achieved at pH 2.8 followed by immediate neutralization ofthe sample. Aggregated protein is separated from monomeric antibodies bysize exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in20 mM Histidine, 150 mM NaCl pH 6.0. Monomeric antibody fractions arepooled, concentrated if required using e.g. a MILLIPORE Amicon Ultra (30MWCO) centrifugal concentrator, frozen and stored at −20° C. Part of thesamples are provided for subsequent protein analytics and analyticalcharacterization e.g. by SDS-PAGE, size exclusion chromatography or massspectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) is used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer is used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography for the determination of the aggregationand oligomeric state of antibodies is performed by HPLC chromatography.Briefly, Protein A purified antibodies are applied to a Tosoh TSKgelG3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5 on an AgilentHPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2×PBS ona Dionex HPLC-System. The eluted protein is quantified by UV absorbanceand integration of peak areas. BioRad Gel Filtration Standard 151-1901served as a standard.

Mass Spectrometry

The total deglycosylated mass of crossover antibodies is determined andconfirmed via electrospray ionization mass spectrometry (ESI-MS).Briefly, 100 μg purified antibodies are deglycosylated with 50 milN-Glycosidase F (PNGaseF, ProZyme) in 100 mM KH₂PO₄/K₂HPO₄, pH 7 at 37°C. for 12-24 h at a protein concentration of up to 2 mg/ml andsubsequently desalted via HPLC on a Sephadex G25 column (GE Healthcare).The mass of the respective heavy and light chains is determined byESI-MS after deglycosylation and reduction. In brief, 50 μg antibody in115 μl are incubated with 60 μl IM TCEP and 50 μl 8 MGuanidine-hydrochloride subsequently desalted. The total mass and themass of the reduced heavy and light chains is determined via ESI-MS on aQ-Star Elite MS system equipped with a NanoMate® source.

EXAMPLES Example 1—Generation of Anti-BCMA Antibodies Example 1A.Production of Antigens and Tool Reagents Example 1A1. Recombinant,Soluble, Human BCMA Extracellular Domain

a) Recombinant, soluble, human BCMA extracellular domain (“BCMA ECD”) isproduced as described in Ryan, 2007 (Mol Cancer Ther; 6 (11): 3009-18).Briefly, the human BCMA extracellular domain (ECD; amino acids 5-51;NP_001183) is amplified with forward primer5-AAGCTTGGATCCATGTTGCAGATGGCTGGGCAGTGCTCC-3 (SEQ ID NO:11) incorporatinga BamH1 site (bold, underlined) and reverse primer5-GAATTCGCGGCCGCTCATCCTTTCACTGAATTGGTCACACTTGCATTAC-3 (SEQ ID NO:12)incorporating a stop codon (italic) and NotI site (bold, underlined)using IMAGE clone 687194 (Invitrogen) as a PCR template. The PCR productis cloned into an expression vector comprising a glutathioneS-transferase gene upstream of glutathione S-transferase (GST),transformed into an E. coli strain comprising T7 RNA polymerase geneunder the control of the lacUV5 promoter and the induced protein ispurified at 4° C. on an ÄKTAexplorer (GE Healthcare). The cell pellet islysed in 1:15 w/v of B-PER buffer (Pierce) containing protease inhibitorand lysozyme. The extract is supplemented with 1 to 2 μg/mL DNase I(Sigma), stirred for an additional 20 min, and adjusted to pH 7.5. Thesoluble fusion protein is collected after centrifugation at 31,000×g for20 min (Beckman) and loaded onto a glutathione Sepharose 4 FF column (GEHealthcare) preequilibrated with B-PER buffer. The column is washed with4 column volumes (CV) B-PER buffer, 3 CV each of wash buffers 1 and 2(Pierce), followed by a final column wash with 5 CV 50 mmol/L Tris (pH8.0), 0.15 mol/L NaCl. The GST-tagged BCMA is eluted with 20 mmol/Lreduced glutathione in 50 mmol/L Tris (pH 8.0) and dialyzed against PBS(pH 7.4) using a 3500 MWCO slide-A-lyzer (Pierce). For GST tag removal,BCMA:GST is treated with thrombin in 50 mmol/L Tris (pH 8.0), 0.15 mol/LNaCl, while bound to the glutathione Sepharose. Released thrombin isthen captured by a benzamidine Sepharose column (GE Healthcare). TheGST-cleaved BCMA is eluted from the column with 3 to 5 CV 50 mmol/L Tris(pH 8.0), 0.15 mol/L NaCl, and dialyzed against PBS (pH 7.4). Thrombinremoval is confirmed by analyzing fractions for thrombin activity usingthe chromogenic substrate S-2238 (Chromogenix, DiaPharma). Proteinconcentration is determined by A280. All purified proteins are analyzedby SDS-PAGE and by TSK-Gel G3000SW HPLC size exclusion chromatography(Tosoh Bioscience).

A biotinylated variant of BCMA ECD (“BCMA-ECD-biot”) is produced asdescribed above using the same procedures with the followingmodifications. A DNA sequence encoding an Avi-His tag is added, via PCRamplification, in frame downstream at the 3′ end of the first PCRproduct described above. This new, second PCR product is then subclonedinto the pGEX4T1 expression vector and then co-transformed in bacteriatogether with a vector for expression of BirA enzyme for in vivobiotinylation of the Avi tag. The remaining production and purificationsteps are performed as indicated above for BCMA-ECD.

b) The extracellular domains of human, cynomolgus and murine BCMA thatwere used as antigens for phage display selections were transientlyexpressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and invivo site-specifically biotinylated via co-expression of BirA biotinligase at the avi-tag recognition sequence located at the C-terminus ofthe Fc portion carrying the receptor chain (Fc knob chain). Theextracellular domains of human, cynomolgus and murine BCMA comprisedmethionine 4 to asparagine 53, methionine 4 to asparagine 52, andalanine 2 to threonine 49, respectively. These were N-terminally fusedto the hinge of a human IgG1 enabling heterodimerization with an unfusedhuman IgG1 Fc portion (hole chain) by knobs-into-holes technology.

Example 1A2. Recombinant, Truncated Murine APRIL

a) Recombinant, truncated murine APRIL is produced as described in Ryan,2007 (Mol Cancer Ther; 6 (11): 3009-18). Briefly, murine APRIL (residues106-241; NP_076006) is amplified from IMAGE clone 5290965 (Invitrogen)and cloned into a bacterial expression vector fused at the COOH terminusto Gene-specific forward primer5-ACGTTAGATCTCCACTCAGTCCTGCATCTTGTTCCAGTTAAC-3 (SEQ ID NO:13) andreverse primer 5-AACGTTGCGGCCGCTAGTTTCACAAACCCCAGG-3 (SEQ ID NO:14) areused for amplification. The BglII and NotI sites (bold, underlined) inthe forward and reverse primers, respectively, are used to clone theresulting PCR fragment a bacterial expression vector fused at the COOHterminus to thioredoxin.thioredoxin The construct is transformed into anEscherichia coli strain K-12 comprising a mutation in the thioredoxinreductase gene and cultured at 25° C. until A600 ˜0.6, induced with 1mmol/L isopropyl-L-thio-β-D-galactopyranoside, and then culturedovernight at 25° C. The E. coli cell paste is resuspended and stirred at4° C. in a 1:10 w/v of B-PER lysis buffer containing complete EDTA-freeprotease inhibitors. The mixture is then diluted with 5× stock buffer toa final concentration of 50 mmol/L Tris-HCl, 0.4 mol/L NaCl, 1%Triton-X100, 5% glycerol, and 10 mmol/L imidazole (pH 8-9). The sampleis supplemented with lysozyme, DNase I, and 2 mmol/L MgCl2 (Sigma),stirred for 30 min, adjusted to 4 mmol/L EDTA, stirred for 20 min, andthen centrifuged to remove the cell debris. The sample is adjusted to 40mmol/L MgCl2 and stirred for 30 min before loading onto a Ni-IMAC column(GE Healthcare). The column is sequentially washed with 3 to 5 CV of 10mmol/L imidazole in 20 mmol/L Tris-HCl (pH 8.0), 2 to 3 CV of 0.5% v/vTriton X-100 in 20 mmol/L Tris-HCl (pH 8.0), then with 5 to 10 CV of 70mmol/L imidazole in 20 mmol/L Tris-HCl (pH 8.0). The truncated-APRIL iseluted with a linear gradient from 70 to 500 mmol/L imidazole in 20mmol/L Tris-HCl, 5% glycerol (pH 8.0). Pooled protein fractions aredialyzed against PBS buffer containing 50 mmol/L imidazole, 0.1 mol/LL-Arg, 5% glycerol, 1 mmol/L EDTA (pH 8.0). The protein concentration isdetermined spectrophotometrically [E280 (1%)=0.94].

b) Recombinant, truncated, murine APRIL that was used as tool(competitor) for the phage display selections and ELISAs was transientlyexpressed as N-terminal monomeric Fc-fusion in HEK EBNA cells. MurineAPRIL comprised histidine 106 to leucine 241. It was N-terminally fusedto the hinge of a human IgG1 enabling heterodimerization with an unfusedhuman IgG1 Fc portion (hole chain) by knobs-into-holes technology.

Example 1A3. Recombinant, Truncated Human BAFF

Recombinant, truncated human is produced as describe in Gordon, 2003(Biochemistry; 42 (20): 5977-5983). Briefly, a DNA fragment encodingBAFF residues 82-285 is cloned into a pBr322 vector comprising a His-Tagat the N-terminus and a subsequent thrombin cleavage site, creating afusion with an N-terminal His-tag followed by a thrombin cleavage site.An E. coli strain comprising T7 RNA polymerase gene under the control ofthe lacUV5 promoter is cultured to mid-log phase at 37° C. in LB mediumwith 50 mg/L carbenicillin and then cooled to 16° C. prior to inductionwith 1.0 mM IPTG. Cells are harvested by centrifugation after 12 h offurther growth and stored at −80° C. The cell pellet is resuspended in50 mM Tris, pH 8.0, and 500 mM NaCl and sonicated on ice. Aftercentrifugation, the supernatant is loaded onto a Ni-NTA agarose column(Qiagen). The column is washed with 50 mM Tris, pH 8.0, 500 mM NaCl, and20 mM imidazole and then eluted with a step gradient in the same bufferwith 250 mM imidazole. BAFF-containing fractions are pooled, thrombin isadded, and the sample is dialyzed overnight against 20 mM Tris, pH 8.0,and 5 mM CaCl₂ at 4° C. The protein is further purified on a monoQ(Pharmacia) column and finally on an S-200 size exclusion column in 20mM Tris, 150 mM NaCl, and 5 mM MgCl₂.

Example 1B. Recombinant Cells Expressing Human BCMA on their Surface

Recombinant cells expressing human BCMA on their surface (“HEK293-BCMAcells”) are generated as described in Ryan, 2007 (Mol Cancer Ther; 6(11): 3009-18). Briefly, full-length human BCMA is amplified usingforward primer 5-GAATTCAAGCTTGCCACCATGTTGCAGATGGCTGGGCAGTGCTCC-3 (SEQ IDNO:15) including a HindIII restriction site (bold, underlined) and Kozakconsensus sequence and reverse primer5-GAATTCTCTAGATTACCTAGCAGAAATTGATTTCTCTATCTCCGTAGC-3 (SEQ ID NO:16)including a 3 stop codon and XbaI restriction site (bold, underlined)using IMAGE clone 687194 (Invitrogen) as a PCR template. Theamplification product is cloned into an E. coli expression vector,comprising human cytomegalovirus (CMV) immediate earlyenhancer/promoter, a polyhistidine (6×His), and a neomycin resistancegene, linearized and transfected into human embryonic kidney 293(HEK293) cells. These cells are selected which express human BCMA ontheir surface high expressing stable clones are chosen byfluorescence-activated cell sorting analysis.

Example 1C. Human Myeloma Cell Line Expressing BCMA on their Surface

a) Cell origin and culture conditions. Human MM cell line NCI-H929 isacquired from the American Type Culture Collection (ATCC CRL-9068).NCI-H929 cells are grown in RPMI 1640 supplemented with 10% fetal calfserum, 2 mM L-Glutamine, 1 mM Sodiumpyruvate. U266B1 (ATCC TIB-196) ahuman B lymphocyte myeloma cell line cultured in RPMI high Glucose, 10%FCS, 1% Glutamine, 1% Sodiumpyruvate, 10 mM HEPES). RPMI 8226 (ATCCCCL-155), a human B lymphocyte myeloma cell line cultured in DMEM, 10%FCS, 1% Glutamine. MKN45 (DSMZ ACC 409), a human gastric adenocarcinomacell line cultured in DMEM containing 10% FCS and 1% Glutamine. BCMAexpression on MM cell lines is confirmed by flow cytometry usingfluorochrome-conjugated anti-human BCMA antibodies (BD Biosciences).

b) BCMA expression was assessed on three human myeloma cell lines (H929,RPMI-8226 and U266B1) by flow cytometry. Briefly, cells were harvested,washed, counted for viability, resuspended at 50 000 cells/well of a96-well round bottom plate and incubated with anti human BCMA antibody(Abcam, #ab54834, mouse IgG1) at 10 μg/ml for 30 min at 4° C. (toprevent internalization). A mouse IgG1 was used as isotype control (BDBiosciences, #554121). Cells were then centrifuged (5 min at 350×g),washed twice and incubated with the FITC-conjugated anti mouse secondaryantibody for 30 min at 4° C. At the end of incubation time, cells werecentrifuged (5 min at 350×g), washed twice with FACS buffer, resuspendedin 100 ul FACS buffer and analyzed on a CantoII device running FACS Divasoftware. FIG. 4 shows increase of median fluorescence intensity uponbinding of increasing concentrations of the anti-BCMA antibody to H929cells. Quantification of BCMA receptor number on membrane surface ofH929, RPMI-8226 and U266B1 myeloma cell lines was assessed by QFIKITanalysis (Dako, #K0078, following manufacturer's instructions).

TABLE 2 Quantification of BCMA receptor number on membrane surface ofH929, RPMI-8226 and U266B1 myeloma cell lines. Myeloma cell lines BCMAreceptor no H929 6085 RPMI-8226 6253 U266(B1) 2865

Example 1D. Obtaining Anti-BCMA Antibodies Via Immunization

Anti-BCMA antibodies are generated by immunization of rats with BCMA ECDas described in Ryan, 2007 (Mol Cancer Ther; 6 (11): 3009-18). Briefly,Sprague-Dawley rats are immunized subcutaneously with keyhole limpethemocyanin-conjugated BCMA ECD (amino acids 5-54; NP_001183) usingTiterMax® adjuvant (Sigma). Keyhole limpet hemocyanin conjugation isperformed with a lysine residue using Imject mcKLHV (Pierce). Due to thehigh sequence homology between human and mouse BCMA proteins, rats arepreferred for antibody production. B cells are harvested from immunizedspleens and fused to P3-X63.Ag8 myeloma cells using a standardpolyethylene glycol fusion protocol (Goding 1996; Monoclonal antibodies:principles and practice. 3^(rd) ed. Academic Press). Hybridomas arecultured in 80% Iscove's modified Dulbecco's medium supplemented with10% fetal clone I, 4 mmol/L L-glutamine, 10% cloning factor and alsoincluding penicillin, streptomycin and 1× sodium hypoxanthine,aminopterin, and thymidine. ELISA testing is performed to detect bindingof hybridoma culture supernatants to BCMA. Positive BCMA-bindinghybridomas are further screened by flow cytometry for cell-based bindingto BCMA transfectants (HEK293-BCMA cells). Chosen hybridomas undergo tworounds of limiting dilution cloning and are further expanded forpurification. In addition, antibodies from those same chosen hybridomasare converted to chimeric antibodies with human constant regions bystandard methods. Briefly, cDNAs encoding the heavy and light chainvariable regions are amplified bz RT-PCR out of mRNA from the hybridomasand then joined in frame with cDNAs coding the heavy constant region ofhuman IgG1 and the human kappa light chain constant region,respectively. These cDNAs are cloned into mammalian transient expressionvectors and plasmid DNA is produced in E. coli and purified fortransfection. HEK293 cells are transfected by a standard transfectionmethod (calcium phosphate-based transfection) and 7 days later IgG1antibodies are purified from culture supernatants by affinitychromatography on a Protein A column followed by isolation of themonomeric antibody fraction via size exclusion chromatography.

Example 1E. Obtaining Anti-BCMA Antibodies Out of an In Vitro,Recombinant Library Example 1E1. Construction of Generic Fab-Libraries

Generic antibody libraries in the Fab-format are constructed on thebasis of human germline genes using the following V-domain pairings:Vk3_20 kappa light chain with VH3_23 heavy chain for the DP47-3 libraryand Vk1_17 kappa light chain with VH1_69 heavy chain for the DP88-3library. Both libraries are randomized in CDR3 of the light chain (L3)and CDR3 of the heavy chain (H3) and are assembled from 3 fragments perlibrary by splicing by overlapping extension (SOE) PCR. Fragment 1comprises the 5′ end of the antibody gene including randomized L3,fragment 2 is a central constant fragment spanning from L3 to H3,whereas fragment 3 comprises randomized H3 and the 3′ portion of theantibody gene. The following primer combinations are used to generatelibrary fragments for DP47-3 library: fragment 1 (LMB3-LibL1b_new),fragment 2 (MS63-MS64), fragment 3 (Lib2H-fdseqlong). See Table 1 ofWO2012020038. The following primer combinations are used to generatelibrary fragments for the DP88-3 library: fragment 1 (LMB3-RJH_LIB3),fragment 2 (RJH31-RJH32) and fragment 3 (LIB88_2-fdseqlong). See tables3 and 4 of WO2012020038.

The PCR protocol for the production of library fragments includes: 5 minof initial denaturation at 94° C.; cycles of 1 min at 94° C., 1 min at58° C., and 1 min at 72° C.; and terminal elongation for 10 min at 72°C. For assembly PCR, equimolar ratios of the 3 fragments are used astemplate. The assembly PCR protocol includes: 3 min of initialdenaturation at 94° C.; and 5 cycles of 30 seconds at 94° C., 1 min at58° C., and 2 min at 72° C. At this stage, primers complementary tosequence outside fragments 1-3 are added and an additional 20 cycles areperformed prior to a terminal elongation for 10 min at 72° C. Afterassembly of sufficient amounts of full length randomized Fab constructs,the Fab constructs are digested with NcoI/NotI for the DP47-3 libraryand with NcoI/NheI for the DP88-3 library alongside with similarlytreated acceptor phagemid vector. For the DP47-3 library, 22.8 μg of Fablibrary is ligated with 16.2 μg of phagemid vector. For the DP88-3library, 30.6 μg of Fab library is ligated with 30.6 μg of phagemidvector.

Purified ligations are used for 68 transformations for the DP47-3library and 64 transformations for the DP88-3 library, respectively, toobtain final DP47-3 and DP88-3 libraries. Phagemid particles displayingthe Fab libraries are rescued and purified by PEG/NaCl purification tobe used for selection of anti-BCMA Fab clones.

Example 1E2. Selection of Anti-BCMA Fab Clones

a) Selections are carried out against BCMA-ECD-biot. The antigen isbiotinylated in vivo upon expression. Selections are carried out insolution according to the following protocol: (i) binding of ⁻10¹²phagemid particles of library DP88-3 and 100 nM BCMA-ECD-biot for 0.5hours in a total volume of 1 ml; (ii) capture of BCMA-ECD-biot andattached phage by the addition of 5.4×10⁷ streptavidin-coated magneticbeads for 10 min; (iii) washing of beads using 5×1 ml PBS/Tween®20 and5×1 ml PBS; (iv) elution of phage particles by the addition of 1 mL 100mM TEA (triethylamine) for 10 min and neutralization by the addition of500 μL 1M Tris/HCl pH 7.4; and (v) re-infection of log-phase E. coli TG1cells (Zymo Research), infection with helper phage VCSM13 (Stratagene)and subsequent PEG/NaCl precipitation of phagemid particles to be usedin subsequent selection rounds.

Selections are carried out over 3 rounds using constant BCMA-ECD-biotconcentrations at 100 nM. In round 2, capture of antigen:phage complexesis performed on neutravidin plates instead of streptavidin beads.Specific binders are identified by ELISA as follows using: 100 μl of 100nM BCMA-ECD-biot is coated in each well of neutravidin plates.

Fab-containing bacterial supernatants are added and binding Fabs aredetected via their Flag-tags by using an anti-Flag/HRP secondaryantibody. Once identified, anti-BCMA ECD clones are bacteriallyexpressed in a 0.5 liter culture volume, affinity purified and furthercharacterized by SPR-analysis using a BIACORE® instrument.

b) Anti-BCMA Fabs were established by phage display from synthetic Fablibraries consisting of VL and VH pairings derived from differentV-domain families. Clones 13C2, 17A5, 83A10, 13D2, 14B11, 14E1, 29B11,and 29F3 were generated from Vk3_20/VH3_23 sublibrary, clone 13A4 fromVk2D_28/VH5_1 sublibrary, and clone 13A7 from Vk2D_28/VH3_23 sublibrary,respectively (Table 3). These libraries are based on entirely humanframeworks with sequence diversity in CDR3 of VL (3 different lengths)and VH domains (6 different lengths).

TABLE 3 Anti-BCMA clones and respective VL/VH pairings Fab clone VLdomain VH domain 13C2 Vk3_20 VH3_23 17A5 Vk3_20 VH3_23 83A10 Vk3_20VH3_23 13A4 Vk2D_28 VH5_1 13D2 Vk3_20 VH3_23 14B11 Vk3_20 VH3_23 14E1Vk3_20 VH3_23 29B11 Vk3_20 VH3_23 29F3 Vk3_20 VH3_23 13A7 Vk2D_28 VH3_23

Selection rounds (biopanning) were performed in solution according tothe following pattern: 1. pre-clearing of ˜10¹² phagemid particles perlibrary pool in immunotubes coated with 10 ug/ml of an unrelated humanIgG to deplete the libraries of antibodies recognizing the Fc-portion ofthe antigens, 2. incubation of the non-Fc-binding phagemid particleswith 100 nM biotinylated BCMA for 0.5 h in the presence of 100 nMunrelated non-biotinylated Fc knobs-into-holes construct for furtherdepletion of Fc-binders in a total volume of 2 ml, 3. capture ofbiotinylated BCMA and specifically binding phage by splitting up andtransferring the panning reaction into 16 wells on a neutravidin orstreptavidin pre-coated microtiter plate for 20 min on a shaker, 4.washing of respective wells 10-30× with PBS/Tween 20 and 10-30× with PBSusing a plate washer, 5. optional competitive washing step by additionof 230 nM murine APRIL to displace Fab clones that recognize the bindingsite of the natural ligand thus selecting for APRIL-non-competing phageantibodies, 6. elution of phage particles by addition of 125 ul 100 mMTEA (triethylamine) per well for 5-10 min and neutralization by additionof an equal volume of 1M Tris/HCl pH 7.4, 7. re-infection of log-phaseE. coli TG1 cells with the eluted phage particles, infection withhelperphage VCSM13, incubation on a shaker at 30° C. overnight andsubsequent PEG/NaCl precipitation of phagemid particles to be used inthe next selection round.

Selections were carried out over 3 to 5 rounds using constant antigenconcentrations of 100 nM. Apart from selection campaigns during whichonly human BCMA was used as antigen, additional selection campaigns werecarried out during which also cynomolgus or murine BCMA were used in analternating fashion with human BCMA in order to select forcross-reactive antibodies. Moreover, as an alternative to streptavidinplate-based capture, capture of antigen: phage complexes was performedby addition of 5.4×107 streptavidin-coated magnetic beads to the panningreaction followed by washing steps using respective magnets under theconditions described above.

Specific binders were identified by surface plasmon resonance-screeningof Fab-containing bacterial culture supernatants using BioRad's ProteOnXPR36 biosensor. In brief, after infection of log-phase E. coli TG1cells with the eluted phage particles, single colony forming units (cfu)were plated and picked for inoculation of 1 ml expression cultures in96-deep well plates. Fabs were captured from the supernatants on aProteOn GLM chip that was derivatized with 8.000-10.000 RU of a goatanti-human IgG, F(ab′)2 fragment specific polyclonal antibody (JacksonImmunoResearch, #109-005-006) in vertical orientation. Subsequently,human, cynomolgus and murine BCMA as well as an unrelated Fcknobs-into-holes construct were injected as analytes in horizontalorientation. Clones that exhibited significant binding responses to BCMAand did not bind the Fc-portion of the antigens, were bacteriallyexpressed in a 0.5 liter culture volume, affinity purified andkinetically characterized by SPR-analysis using a one-shot-kineticsprotocol on BioRad's ProteOn XPR36 biosensor.

Example 1F. BCMA Binding Assays: Surface Plasmon Resonance

a) To measure binding affinities of BCMA antibody to immobilized BCMA,surface plasmon resonance measurements are performed on a Biacore® 3000instrument (Pharmacia Biosensor). The receptor BCMA (BCMA-ECD) iscoupled to the sensor chip at a level of 400 resonance units using theamine coupling protocol as provided by manufacturer. AlternativeBCMA-ECD-biot is coupled to a streptavidin-sensor chip, also at a levelof 400 resonance units, using the protocol as provided by themanufacturer. In all experiments, flow cell 1 is used as the referencecell. Sensorgrams are recorded for Fab solutions ranging inconcentration from 0.1 pM to 200 nM. Nonlinear regression analysis isused to calculate kinetic constants and binding constants simultaneouslywith the use of the manufacturer's software. Fab clones with monovalentbinding affinities to BCMA-ECD of 100 nM are converted into IgGs bystandard methods. Briefly, cDNAs encoding the heavy and light chainvariable regions are joined in frame with cDNAs coding the heavyconstant region of human IgG1 and the human kappa light chain constantregion, respectively. These cDNAs are cloned into mammalian transientexpression vectors and plasmid DNA is produced in E. coli and purifiedfor transfection. HEK293 cells are transfected by a standardtransfection method (calcium phosphate-based transfection) and 7 dayslater IgG1 antibodies are purified from culture supernatants by affinitychromatography on a Protein A column followed by isolation of themonomeric antibody fraction via size exclusion chromatography.

b) Affinities (KD) of anti-BCMA Fab clones were measured by surfaceplasmon resonance using a ProteOn XPR36 instrument (Biorad) at 25° C.with biotinylated human, cynomolgus and murine BCMA immobilized on NLCchips by neutravidin capture (Table 4). An unrelated biotinylated Fcknobs-into-holes construct was immobilized in a similar fashion asnegative control Immobilization of antigens (ligand): Recombinantantigens were diluted with PBST (10 mM phosphate, 150 mM sodium chloridepH 7.4, 0.005% Tween 20) to 10 ug/ml, then injected at 40 ul/minute for300 s in vertical orientation. Injection of analytes: For one-shotkinetics measurements, injection direction was changed to horizontalorientation, two-fold dilution series of purified Fab (varyingconcentration ranges) were injected simultaneously at 40 ul/min alongchannels 1-5, with association times of 200 or 300 s, and dissociationtimes of 300 s. Buffer (PBST) was injected along the sixth channel toprovide an “in-line” blank for referencing. Association rate constants(kon) and dissociation rate constants (koff) were calculated using asimple one-to-one Langmuir binding model in ProteOn Manager v3.1software by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (KD) was calculatedas the ratio koff/kon. Regeneration was performed in horizontalorientation using 10 mM glycine-HCl pH 1.5 at a flow rate of 100 ul/minfor a contact time of 18 s.

TABLE 4 Monovalent affinities of anti-BCMA Fab clones: Fab K_(D) humanK_(D) cynomolgus K_(D) murine clone BCMA [nM] BCMA [nM] BCMA [nM] 13C2196 — 144 17A5 45 — 74 83A10 76 1510 1134 13A4 1.8 — — 13D2 86 weak weak14B11 383 — — 14E1 91 weak weak 29B11 224 — weak 29F3 87 — weak 13A7 235— —

c) Assessment of binding of anti-BCMA antibodies to recombinant BCMA bysurface plasmon resonance (SPR) as follow. All SPR experiments wereperformed on a Biacore T200 at 25° C. with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20,Biacore, Freiburg/Germany). The avidity of the interaction betweenanti-BCMA antibodies and recombinant BCMA Fc(kih) (human, cynomolgus andmurine) was determined. Biotinylated recombinant human, cynomolgus andmurine BCMA Fc(kih) were directly coupled on a SA chip followinginstructions (Biacore, Freiburg/Germany). The immobilization levelranged from 200 to 700 RU. The anti-BCMA antibodies were passed at a2-fold concentration range (1.95 to 500 nM) with a flow of 30 μL/minutesthrough the flow cells over 120 seconds. The dissociation was monitoredfor 180 seconds. Bulk refractive index differences were corrected for bysubtracting the response obtained on the reference flow cell. Here, theanti-BCMA antibodies were flown over an empty surface previouslyactivated and deactivated as described in the standard amine couplingkit. Apparent kinetic constants were derived using the Biacore T200Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rateequations for 1:1 Langmuir binding by numerical integration, despite thebivalency of the interaction for comparison purposes.

The affinity of the interaction between anti-BCMA antibodies andrecombinant human BCMA Fc(kih) was also determined. Anti-human Fabantibody (GE Healthcare) was directly coupled on a CM5 chip at pH 5.0using the standard amine coupling kit (Biacore, Freiburg/Germany). Theimmobilization level was about 6500 RU. Anti-BCMA antibody was capturedfor 90 seconds at 25 nM. Recombinant human BCMA Fc(kih) was passed at a4-fold concentration range (1.95 to 500 nM) with a flow of 30 μL/minutesthrough the flow cells over 120 seconds. The dissociation was monitoredfor 120 seconds. Bulk refractive index differences were corrected for bysubtracting the response obtained on reference flow cell. Here,recombinant BCMA was flown over a surface with immobilized anti-humanFab antibody but on which HBS-EP has been injected rather than anti-BCMAantibody. Kinetic constants were derived using the Biacore T100Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rateequations for 1:1 Langmuir binding by numerical integration (Table 5).Binding of 83A10 anti-BCMA antibody to recombinant cynomolgus BCMAFc(kih) and murine BCMA Fc(kih) was also measured (Table 6).

TABLE 5 Affinity constants determined by fitting rate equations for 1:1Langmuir binding Kon Koff KD Ligand Analyte (1/Ms) (1/s) (M) 13C2anti-BCMA IgG huBCMA Fc(kih) 2.4E+05 1.1E−02 4.7E−08 17A5 anti-BCMA IgGhuBCMA Fc(kih) 2.2E+05 1.9E−03 8.7E−09 83A10 anti-BCMA IgG huBCMAFc(kih) 6.2E+05 2.5E−03 4.1E−09 29F3 anti-BCMA IgG huBCMA Fc(kih)3.2E+05 6.8E−03 2.1E−08 13A7 anti-BCMA IgG huBCMA Fc(kih) 8.0E+047.9E−03 1.0E−07 13A4 anti-BCMA IgG huBCMA Fc(kih) 7.2E+04 3.6E−045.1E−09 13D2 anti-BCMA IgG huBCMA Fc(kih) 3.6E+05 9.3E−03 2.6E−08 14B11anti-BCMA IgG huBCMA Fc(kih) 1.5E+05 1.6E−02 1.1E−07 14E1 anti-BCMA IgGhuBCMA Fc(kih) 4.0E+05 8.1E−03 2.0E−08 29B11 anti-BCMA IgG huBCMAFc(kih) 1.7E+05 6.6E−03 4.0E−08

TABLE 6 Binding of recombinant BCMA Fc(kih) to 83A10 anti-BCMA antibody.Kon Koff KD Ligand Analyte (1/Ms) (1/s) (M) 83A10 anti-BCMA IgG huBCMAFc(kih) 6.2E+05 2.5E−03 4.1E−09 83A10 anti-BCMA IgG cyBCMA Fc(kih)2.8E+05 2.0E−02 7.2E−08 83A10 anti-BCMA IgG muBCMA Fc(kih) 2.0E+054.0E−02 2.0E−07 a) cynomolgus BCMA Fc(kih). b) murine BCMA Fc(kih)

Example 1G. BCMA-Signaling Assay: NF-κB Activation

a) As described in Ryan, 2007 (Mol Cancer Ther; 6 (11): 3009-18),NCI-H929 cells are washed and incubated in RPMI supplemented with 0.25%fetal bovine serum for 24 h before treatment. The cells are thenuntreated or treated with 0.1 μg/mL TNF-α, 100 ng/mL, preferably 1000ng/mL heat-treated HT-truncated-APRIL, 100 ng/mL, preferably 1000 ng/mLtruncated-APRIL, 0.1 pM to 200 nM isotype control, or 0.1 pM to 200 nManti-BCMA antibodies for 20 min. To evaluate ligand blockade, cellspre-treated for 20 min with 0.1 pM to 200 nM of anti-BCMA antibodies oran isotype control antibody are treated with 1 μg/mL of truncated-APRIL.Cells are then harvested, washed, and lysed with 50 mmol/L Tris-HCl (pH7.5), 1% NP₄0, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTAsupplemented with protease, and phosphatase inhibitors. Protein extractsare then analyzed for NF-κB activity using a TransAM® chemiluminescentassay kit (Active Motif) and the luminescent signal reading is performedwith a Fusion HT plate reader (Packard Instruments).

b) Briefly, H929 cells were starved in RPMI 1640 with 0.25% FCS for 24 hat 37° C. in cell incubator. At the end of the starvation time, cellswere harvested, counted and cell viability evaluated using ViCell.Viable cells were adjusted to 4×106 cells per ml in BSA-containing FACSStain Buffer (BD Biosciences). 30 μl of this cell suspension werefurther aliquoted per well into a round-bottom 96-well plate andpre-incubated with anti-BCMA antibodies (15 or 50 ug/ml) or isotypecontrol antibodies (10, and 40 ug/ml) for 20 min in cell incubator.Cells were then supplemented with 1 ug/ml recombinant mouse Δ-APRILtagged with hemagglutinin (HA) (R&D Systems Europe, #7907-AP-010) for 40min at 37° C. Heat inactivated Δ-APRIL (HI APRIL) was used in the assayto confirm the specificity of Δ-APRIL-induced NFkB signal (heatinactivation was performed by treatment of Δ-APRIL at 60° C. for 1 h).At the end of incubation time, cells were harvested, washed, lysed, andprocessed according to the manufacturer's protocol of the NuclearExtract Kit (Active Motif, #40410). Protein extracts were analyzed forNF-κB activity using a TransAm© NFκB p65 Chemi Assay kit (Active Motif,#40097) following manufacturer's instructions. Luminescent signal wasread using the Spectra Max M5 luminometer (Molecular Devices).

Example 1H. Screening Anti-BCMA Antibodies to Select Antibodies notAffected by 100 ng/mL, Preferably 1000 ng/mL of APRIL or BAFF in theirBinding to BCMA and that Neither Promote Nor Block Signaling Via theBCMA Intracellular Domain

The invention relates to the generation of an anti-human BCMA antibodythat 1) binds to human BCMA, 2) binding to BCMA is not affected by 100ng/mL, preferably 1000 ng/mL of APRIL and BAFF, 3) does not block orreduce >20%, preferably >15% or increase >20%, preferably >15%APRIL-dependent NF-κB activation, 4) does not block or reduce >20%,preferably >15% or increase >20%, preferably >15% BAFF-dependent NF-κBactivation, 5) does not induce NF-κB activation by itself, without APRILor BAFF. Table 10 shows the screening paradigm for selection of a BCMAantibody with desired new properties: non-ligand binding/blocking,non-ligand competing. Antibodies are selected whose binding to BCMA isnot blocked by APRIL or by BAFF.

Example 1H1. Binding to BCMA on HEK293-BCMA Cells, Plate-Bound-BCMA orBCMA-Positive Multiple Myeloma Cell Lines (Flow Cytometry and ELISA)

a) Anti-BCMA antibodies coming either from the immunization approachand/or from the screening of the recombinant in vitro library describedabove are analyzed by flow cytometry for binding to human BCMA onHEK293-BCMA cells. Briefly, cultured cells are harvested, counted andcell viability is evaluated using the Trypan Blue exclusion method.Viable cells are then adjusted to 2×10⁶ cells per ml in BSA-containingFACS Stain Buffer (BD Biosciences). 90 μl of this cell suspension arefurther aliquoted per well into a round-bottom 96-well plate. 10 μl ofthe anti-BCMA antibodies or corresponding IgG control are added to thecell-containing wells to obtain final concentrations of 0.1 pM to 200nM. All constructs and control IgG are used at the same molarity. Afterincubation for 30 min at 4° C., the cells are centrifuged (5 min,350×g), washed with 150 μl/well FACS Stain Buffer (BD Biosciences),resuspended and incubated for an additional 30 min at 4° C. with 12μl/well fluorochrome-conjugated AffiniPure F(ab′)2 Fragment goatanti-human IgG Fcγ Fragment Specific (Jackson Immuno Research Lab;working solution: 1:20). Cells are then washed with Stain Buffer (BDBiosciences) 120 μl/well and pelleted down by centrifugation at 350×gfor 5 min. A second washing step is performed using FACS Stain Buffer150 μl/well. The samples are resuspended in 200 μl/well FACS StainBuffer and acquired and analyzed using an LSR II flow cytometer withFACSDiva® software (BD Biosciences). The mean fluorescence intensity(MFI) is plotted as a function of anti-BCMA antibody concentration toobtain the binding curve and to calculate the effective antibodyconcentration to reach 50% of maximal binding (EC₅₀). Anti-BCMAantibodies that bind to BCMA on cells as judged from this assay areselected for the next screening step, namely the competition BCMAbinding assay against APRIL and BAFF (step (Example 1H2) below).

The properties of antibodies that show binding to human BCMA onHEK293-BCMA cells are confirmed using an ELISA method as described byRyan et al. (2007). Briefly, immunosorb 96-well plates are coated with1.5 μg/mL of GST-BCMA-ECD, washed with PBS+1% Tween (PBS-T), and blockedwith PBS-T plus 1% serum albumin BCMA-coated plates are incubated withhybridoma culture supernatants for 2 h at room temperature, washed 5times with PBS-T, and incubated with peroxidase-conjugated goat-anti-ratIgG. Following incubation with secondary antibody, plates are washed,incubated with 3,3,5,5-tetramethylbenzidine substrate, and stopped withan equal volume of 1 mol/L H₂SO₄.

b) Anti-BCMA IgG antibodies (clones 13C2, 17A5, 83A10, 13A4, 13D2, 14E1,13A7, 14B11) were analyzed by flow cytometry for binding to human BCMAon BCMA-expressing H929 cells. MKN45 (human gastric adenocarcinoma cellline that does not express BCMA) was used as negative control. Briefly,cultured cells are harvested, counted and cell viability was evaluatedusing ViCell. Viable cells are then adjusted to 2×10⁶ cells per ml inBSA-containing FACS Stain Buffer (BD Biosciences). 100 μl of this cellsuspension were further aliquoted per well into a round-bottom 96-wellplate and incubated with 30 μl of the anti-BCMA antibodies orcorresponding IgG control for 30 min at 4° C. All anti-BCMA antibodies(and isotype control) were titrated and analyzed in final concentrationrange between 0.1-40 ug/ml. Cells were then centrifuged (5 min, 350×g),washed with 120 μl/well FACS Stain Buffer (BD Biosciences), resuspendedand incubated for an additional 30 min at 4° C. withfluorochrome-conjugated PE-conjugated AffiniPure F(ab′)2 Fragment goatanti-human IgG Fc Fragment Specific (Jackson Immuno Research Lab;109-116-170). Cells were then washed twice with Stain Buffer (BDBiosciences), fixed using 100 ul BD Fixation buffer per well (#BDBiosciences, 554655) at 4° C. for 20 min, resuspended in 120 μl FACSbuffer and analyzed using BD FACS CantoII. FIG. 5 shows the meanfluorescence intensity for anti-BCMA IgG clones plotted in function ofanti-BCMA antibody concentration; (A) clones 13C2, 17A5, 83A10 on H929cells, (B) clones 13C2, 17A5, 83A10 on MKN45 cells, (C) clones 13A4,13D2, 14E1, 13A7, 14B11 on H929 cells (D) clones 13A4, 13D2, 14E1, 13A7,14B11 on MKN45 cells. EC50 values (denoting the antibody concentrationrequired to reach 50% of the maximal binding) for the binding of clones13C2, 17A5, 83A10 to H929 cells are summarized in Table 7.

TABLE 7 EC50 values for binding of anti-BCMA antibodies to H929 multiplemyeloma cells Anti-BCMA Anti-BCMA Anti-BCMA antibody clone antibodyclone antibody clone 13C2 83A10 17A5 EC50 (nM) 13.9 12.5 9.0 EC50(ug/ml) 2.0 1.8 1.3

Example 1H2. 100 ng/mL, Preferably 1000 ng/mL of APRIL or BAFF does notAlter BCMA Antibody Binding to Human-BCMA (Flow Cytometry and ELISA)

a) Anti-BCMA antibodies selected from step (Example 1H1) above are thenanalyzed by flow cytometry for binding to human BCMA on HEK293-BCMAcells in the presence and absence of 100 ng/mL, preferably 1000 ng/mLAPRIL or BAFF. Viable HEK293-BCMA cells are adjusted to 2×10⁶ cells perml in BSA-containing FACS Stain Buffer (BD Biosciences). 90 μl of thiscell suspension are further aliquoted per well into a round-bottom96-well plate. 10 μl of the anti-BCMA antibodies or corresponding IgGcontrol are added to the cell-containing wells to obtain finalconcentrations of 0.1 pM to 200 nM. All constructs and control IgG areused at the same molarity. After incubation for 30 min at 37° C., in thepresence and absence of 100 ng/ml, preferably 1000 ng/mL of APRIL andBAFF, respectively, the cells are centrifuged (5 min, 350×g), washedwith 150 μl/well FACS Stain Buffer (BD Biosciences), resuspended andincubated for an additional 30 min at 4° C. with 12 μl/wellfluorochrome-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgGFcγ Fragment Specific (Jackson Immuno Research Lab; working solution:1:20). Cells are then washed with Stain Buffer (BD Biosciences) 120μl/well and pelleted down by centrifugation at 350×g for 5 min. A secondwashing step is performed using FACS Stain Buffer 150 μl/well. Thesamples are resuspended in 200 μl/well FACS Stain Buffer and acquiredand analyzed using an LSR II flow cytometer with FACSDiva® software (BDBiosciences). The mean fluorescence intensity is plotted as a functionof anti-BCMA antibody concentration to obtain the binding curve and tocalculate the effective antibody concentration to reach 50% of maximalbinding (EC₅₀). One binding curve is done in the presence of APRIL,another in its absence, and the same is done for presence and absence ofBAFF. Those antibodies whose binding to BCMA is not affected by 100ng/ml, preferably 1000 ng/mL of APRIL and also is not affected by 100ng/ml, preferably 1000 ng/mL of BAFF are selected for next steps below.Representative binding curves for antibodies that are non-competing withthe ligands APRIL and BAFF for binding to BCMA and for antibodies thatare competing with these ligands for binding to BCMA are shown in FIG.1.

The properties of antibodies that show binding to human BCMA onHEK293-BCMA cells in the presence of 100 ng/mL, preferably 1000 ng/mLAPRIL or BAFF are confirmed using an ELISA method as described by Ryanet al. (2007). Briefly, immunosorb 96-well plates are coated with 1.5μg/mL of GST-BCMA-ECD, washed with PBS+1% Tween® (PBS-T), and blockedwith PBS-T plus 1% serum albumin BCMA-coated plates are incubated withhybridoma culture supernatants for 2 h at room temperature, washed 5times with PBS-T, and incubated with peroxidase-conjugated goat-anti-ratIgG. Following incubation with secondary antibody, plates are washed,incubated with 3,3,5,5-tetramethylbenzidine substrate, and stopped withan equal volume of 1 mol/L H₂SO₄. For plate-based ligand blockade,plates are coated with 1 μg/mL of GST-BCMA-ECD as described above.Coated plates are preincubated with purified antibodies at the specifiedconcentrations, washed with PBS-T, and then incubated with 3 μg/mL ofrecombinant human MegaAPRIL (Alexis Biochemicals) or recombinant humanBAFF (R&D Systems). APRIL or BAFF binding is detected usingperoxidase-conjugated anti-FLAG followed by development with3,3′,5,5′-tetramethylbenzidine as described above.

b) Identification of non-APRIL-competing anti-BCMA Fabs or antibodies byELISA. Binding of Fabs to immobilized human BCMA was assessed in thepresence of increasing concentrations of murine APRIL. 25 nMbiotinylated human BCMA (100 μl/well) were coated on a neutravidin plateand incubated on a shaker for 1 h at room temperature. 500 nM or 1000 nMpurified Fabs were added to saturate the coated human BCMA for 1 h atroom temperature. The plate was washed 3 times with PBS and murine APRILwas added at eight different concentrations using a two-fold dilutionseries in PBS buffer, ranging from 0 to 100 nM, and incubated on ashaker for 30 min. The plate was washed 3 times with PBS andanti-FLAG-HRP secondary antibody (1:4000) was added for 1 h. Again, theplate was washed 3 times with PBS and developed by adding 100 μl/well BMBlue POD (Roche). The reaction was stopped by adding 50 ul/well 1M H₂SO₄and the OD was read at 450 nm (reference at 650 nm) for a final read-outof OD₄₅₀₋₆₅₀. Results for selected Fabs are shown in FIG. 6. Thereduction (%) in OD values measured with the anti-BCMA clones in theabsence vs. presence of 50 nM (1000 ng/mL) or 6.25 nM (140 ng/mL)muAPRIL (murine APRIL) is summarized in Table 8.

TABLE 8 Reduction in OD values measured (450 nm) in absence vs. presenceof muAPRIL Reduction (↓) in OD values in presence of muAPRIL muAPRILAnti-BCMA antibody clones (nM and ng/mL) 13C2 17A5 83A10 13A4 13D2 29B1113A7   50 nM/1000 ng/mL 18.9% 34.5% 6.3% 13.1% 7.3% 67.3% 93.2% 6.25nM/140 ng/mL no ↓ 5.6% no ↓ 7.7% 6.4% 12.1% 31.3%

c) Competition of Δ-APRIL with anti-BCMA antibodies detected by flowcytometry. The assessment of the eventual competition between Δ-APRILand anti-BCMA antibodies was performed on H929 cells by quantifying thebinding of Δ-APRIL in presence of increasing concentrations of anti-BCMAantibodies (clones 13C2, 17A5, 83A10, 13A4, 13D2, 14E1, 13A7, 14B11).Briefly, cultured cells were harvested, counted and cell viabilityevaluated using ViCell. Viable cells were adjusted to 1×10⁶ cells per mlin BSA-containing FACS Stain Buffer (BD Biosciences). 100 μl of thiscell suspension are further aliquoted per well into a round-bottom96-well plate and incubated with 30 μl of the anti-BCMA antibodies orcorresponding IgG control for 30 min at 4° C. All anti-BCMA antibodies(and isotype control) are titrated and analyzed at final concentrationsof 1, 16 and 40 ug/ml. Cells are then centrifuged (5 min, 350×g), washedwith 120 μl/well FACS Stain Buffer (BD Biosciences), resuspended andincubated with 1 ug/ml recombinant mouse Δ-APRIL tagged withhemagglutinin (HA) (R&D Systems Europe, #7907-AP-010) for additional 30min at 4° C. Cells are then washed once with 120 μl/well FACS Buffer andincubated with FITC-conjugated anti-HA antibody (Sigma Aldrich, #H7411)for 30 min at 4° C. At the end of incubation time, cells are washed with120 μl/well FACS Buffer, fixed using 100 ul BD Fixation buffer per well(#BD Biosciences, 554655) at 4° C. for 20 min, resuspended in 80 μl FACSbuffer and analyzed using BD FACS Fortessa. FIG. 7 shows the relativemedian fluorescence intensity of Δ-APRIL (FITC signal) detected infunction of increasing concentrations of anti-BCMA antibody clones 13A4,13D2, 14E1, 13A7, 14B11 on H929 cells. The median fluorescence intensityupon binding of Δ-APRIL in presence of the isotype control was set toone; the other signals were normalized to it.

d) Competition of anti-BCMA antibodies with Δ-APRIL detected by flowcytometry. The assessment of the eventual competition between Δ-APRILand anti-BCMA antibodies was performed on RPMI cells by quantifying thebinding of anti-BCMA antibodies (clones 13A4, 13C2, 13D2, 14B11, 17A5,83A10,) in presence or absence of Δ-APRIL. Briefly, cultured cells wereharvested, counted and cell viability evaluated using ViCell. Viablecells were adjusted to 1×10⁶ cells per ml in BSA-containing FACS StainBuffer (BD Biosciences). 100 μl of this cell suspension were furtheraliquoted per well into a round-bottom 96-well plate and incubated with30 μl of the anti-BCMA antibodies or corresponding IgG control for 20min at 4° C. All anti-BCMA antibodies and isotype control were analyzedat final concentrations 40 ug/ml. Cells were then centrifuged (5 min,350×g), washed with 120 μl/well FACS Stain Buffer (BD Biosciences),resuspended and incubated with 1 ug/ml recombinant mouse Δ-APRIL taggedwith hemagglutinin (HA) (R&D Systems Europe, #7907-AP-010) foradditional 40 min at 4° C. Cells were then washed once with 120 μl/wellFACS Buffer and incubated with with Alexa.Fluor 647-conjugatedanti-human Fc antibody (Jackson Immuno Research Lab, #109-606-008) for30 min at 4° C. At the end of incubation time, cells were washed with120 μl/well FACS Buffer, fixed using 100 ul BD Fixation buffer per well(#BD Biosciences, 554655) at 4° C. for 20 min, resuspended in 80 μl FACSbuffer and analyzed using BD FACS Fortessa. FIG. 8 shows the relativemedian fluorescence intensity of anti-BCMA antibody (Alexa.Fluor 647signal) clones 13A4, 13C7, 13D2, 14B11, 17A5, 83A10 on RPMI cellsdetected in absence or presence of 1000 ng/mL of Δ-APRIL. The medianfluorescence intensity upon binding of anti-BCMA antibodies in absenceof Δ-APRIL was set to one; the other signals respective to the anti-BCMAantibody in presence of Δ-APRIL were normalized to it.

e) Competition of anti-BCMA antibodies with Δ-APRIL after simultaneousincubation detected by flow cytometry. The assessment of the eventualcompetition between Δ-APRIL and anti-BCMA antibodies was performed onH292 cells (NCI-H929, ATCC® CRL9068™) by quantifying the binding ofanti-BCMA antibodies (clones 14B11, 13D2, 13A4, 17A5, 83A10) in presenceor absence of Δ-APRIL. Briefly, cultured cells were harvested, countedand cell viability evaluated using ViCell. Viable cells were adjusted to1×10⁶ cells per ml in BSA-containing FACS Stain Buffer (BD Biosciences).100 μl of this cell suspension were further aliquoted per well into around-bottom 96-well plate and incubated with 30 μl of the anti-BCMAantibodies or corresponding IgG control and 30 μl of Δ-APRIL tagged withhemagglutinin (HA) (R&D Systems Europe, #7907-AP-010) for 40 min at 4°C. All anti-BCMA antibodies and isotype control were analyzed at finalconcentrations 20 ug/ml; Δ-APRIL at final concentrations 2.5 ug/ml.Cells were then centrifuged (5 min, 350×g) and washed with 120 μl/wellFACS Stain Buffer (BD Biosciences). After that, cells were incubatedwith with Alexa.Fluor 647-conjugated anti-human Fc antibody (JacksonImmuno Research Lab, #109-606-008) and FITC-conjugated anti-HA antibody(Sigma Aldrich, #H7411) for 30 min at 4° C. At the end of incubationtime, cells were washed with 120 μl/well FACS Buffer, fixed using 100 ulBD Fixation buffer per well (#BD Biosciences, 554655) at 4° C. for 20min, resuspended in 80 μl FACS buffer and analyzed using BD FACSCantoII. FIG. 9A shows the mean fluorescence intensity and the relativefluorescence signal of the anti-BCMA antibody clone (Alexa.Fluor 647signal) and FIG. 9B shows the mean fluorescence intensity and therelative fluorescence signal of Δ-APRIL (FITC signal) and the anti-BCMAantibody clone (Alexa.Fluor 647 signal). Detection of anti-BCMA antibodyin presence of Δ-APRIL with FITC-conjugated anti-human Fc antibody wasnormalized to the signal of anti-BCMA antibody clone in absence Δ-APRIL.Detection of Δ-APRIL in presence of the anti-BCMA antibody clone withAlexa.Fluor 647-conjugated anti-HA antibody was normalized to Δ-APRILsignal in presence of the isotype control. Reduction in binding ofanti-BCMA antibodies (20 μg/mL) clones 14B11, 13D2, 13A4, 17A5 and 83A10in presence of Δ-APRIL (2.5 μg/mL) as detected withfluorochrome-conjugated anti-human Fc antibody is summarized in Table 9.

TABLE 9 Reduction in binding of anti-BCMA antibodies to H929 cellsinpresence of APRIL Anti-BCMA Reduction (↓) in binding of anti-BCMAantibody clones antibodies in presence of APRIL 14B11 50% 13D2 25% 13A425% 17A5 20% 83A10 10%

Example 1H3. BCMA Antibody does not Block or Increase APRIL-DependentNF-κB Activation

a) Antibodies selected as non-competing in step (Example 1H2) above(i.e., their BCMA binding curve is not affected by the presence of 100ng/ml, preferably 1000 ng/mL of APRIL and is also not affected by thepresence of 100 ng/ml, preferably 1000 ng/mL of BAFF) are then tested instep (Example 1H3) for effects on APRIL, BAFF, and BCMA mediated NF-κBactivation. As APRIL is the high affinity ligand to BCMA, the blockingor agonist properties of anti-BCMA antibodies on APRIL signaling isfirst examined. As described in Ryan 2007 (Mol Cancer Ther; 6 (11):3009-18), to verify whether anti-BCMA antibodies block or increase APRILdownstream signaling, NCI-H929 human multiple myeloma (MM) cells arewashed and incubated in serum free RPMI for 24 h before treatment. Thecells are then untreated or treated with 0.1 μg/mL TNF-α (used aspositive control), 100 ng/mL, preferably 1000 ng/mL heat-treatedHT-truncated-APRIL, 100 ng/mL, preferably 1000 ng/mL truncated-APRIL,0.1 pM to 200 nM isotype control, or 0.1 pM to 200 nM anti-BCMAantibodies for 20 min. To evaluate APRIL blockade, cells pre-treated for20 min with 0.1 pM to 200 nM of anti-BCMA antibodies or a respectiveisotype control antibody are treated with 100 ng/mL, preferably 1000ng/mL of truncated-APRIL. Cells are then harvested, washed, and lysedwith 50 mmol/L Tris-HCl (pH 7.5), 1% NPκ0, 150 mmol/L NaCl, 1 mmol/LEDTA, 1 mmol/L EGTA supplemented with protease, and phosphataseinhibitors. Protein extracts are then analyzed for NF-κB activity usinga TransAM® chemiluminescent assay kit (Active Motif) and the luminescentsignal reading is performed with a Fusion HT plate reader (PackardInstruments).

As NF-κB activity is assayed using a functional ELISA that detectschemiluminescent signal from p65 bound to the NF-κB consensus sequence,anti-BCMA antibodies that do not alter APRIL-mediated downstreamsignaling and NF-κB activation (i.e. that the mean luminescent signaldetected by ELISA in nuclear extracts from NCI-H929 MM cells treatedwith APRIL alone is similar, not significantly reduced or increased, tothat of nuclear extracts from NCI-H929 MM cells treated with APRIL andanti-BCMA antibodies) are selected for the next steps below.

b) It was assessed whether binding of anti-BCMA antibodies interfereswith APRIL-induced NFkB activation, a known signaling pathway downstreamof BCMA. Briefly, H929 cells were starved in RPMI 1640 with 0.25% FCSfor 24 h at 37° C. in cell incubator. At the end of the starvation time,cells were harvested, counted and cell viability evaluated using ViCell.Viable cells were adjusted to 4×10⁶ cells per ml in BSA-containing FACSStain Buffer (BD Biosciences). 30 μl of this cell suspension werefurther aliquoted per well into a round-bottom 96-well plate andpre-incubated with anti-BCMA antibodies (15 or 50 ug/ml) or isotypecontrol antibodies (10, 20 and 40 ug/ml) for 20 min in cell incubator.Cells were then supplemented with 1 ug/ml recombinant mouse Δ-APRILtagged with hemagglutinin (HA) (R&D Systems Europe, #7907-AP-010) for 40min at 37° C. Heat inactivated Δ-APRIL (HI APRIL) was used in the assayto confirm the specificity of Δ-APRIL-induced NFkB signal (heatinactivation was performed by treatment of Δ-APRIL at 60° C. for 1 h).At the end of incubation time, cells were harvested, washed, lysed, andprocessed according to the manufacturer's protocol of the NuclearExtract Kit (Active Motif, #40410). Protein extracts were analyzed forNF-kB activity using a TransAm© NfkB p65 Chemi Assay kit (Active Motif,#40097) following manufacturer's instructions. Luminescent signal wasread using the Spectra Max M5 luminometer (Molecular Devices). Therelative luminescence signal intensity obtained using H929 cells treatedas described above was measured. The luminescence signal obtained uponbinding of Δ-APRIL in presence of the isotype control was set to one;the other signals were normalized to it.

Example 1H4. BCMA Antibody does not Block or Increase BAFF-DependentNF-κB Activation

Antibodies selected as non-blocking and non-increasing APRIL-dependentNF-κB activation in step (Example 1H3) above are then tested in step(Example 1H3) for effects on BAFF mediated NF-κB activation. Asdescribed in Ryan 2007, (Mol Cancer Ther; 6 (11): 3009-18), to verifywhether BCMA antibodies block or increase BAFF downstream signalingleading to NF-κB activation, NCI-H929 MM cells (CRL9068™) are washed andincubated in serum free RPMI medium for 24 h before treatment, asdescribed in Ryan 2007, (Mol Cancer Ther; 6 (11): 3009-18), The cellsare then untreated or treated with 0.1 μg/mL TNF-α, 100 ng/mL,preferably 1000 ng/mL heat-treated HT-truncated-BAFF, 100 ng/mL,preferably 1000 ng/mL truncated-BAFF, 0.1 pM to 200 nM isotype control,or 0.1 pM to 200 nM anti-BCMA antibodies for 20 min. To evaluate BAFFblockade, cells pre-treated for 20 min with 0.1 pM to 200 nM ofanti-BCMA antibodies or a respective isotype control antibody aretreated with 1 μg/mL of truncated-BAFF. Cells are then harvested,washed, and lysed with 50 mmol/L Tris-HCl (pH 7.5), 1% NP₄0, 150 mmol/LNaCl, 1 mmol/L EDTA, 1 mmol/L EGTA supplemented with protease, andphosphatase inhibitors. Protein extracts are then analyzed for NF-κBactivity using a TransAM® chemiluminescent assay kit (Active Motif) andthe luminescent signal reading is performed with a Fusion HT platereader (Packard Instruments). Anti-BCMA antibodies that do not alterBAFF-mediated downstream signaling and NF-κB activation (i.e. that themean luminescent signal from p65 bound to the NF-κB consensus sequencedetected by ELISA in nuclear extracts from NCI-H929 MM cells treatedwith BAFF alone is similar, not significantly reduced or increased, tothat of nuclear extracts from NCI-H929 MM cells treated with BAFF andanti-BCMA antibodies) are selected for the next steps below.

Example 1H5. BCMA Antibody does not Induce NF-κB Activation by Itself

a) Antibodies selected as non-blocking and non-increasing BAFF-dependentNF-κB activation in step (Example 1H4) above are then tested in step(Example 1H5) for their intrinsic agonistic effects to mediate NF-κBactivation. To verify whether BCMA antibodies are agonistic and inducedownstream signaling by themselves, NCI-H929 cells are washed andincubated in serum-free RPMI medium for 24 h before treatment. The cellsare then untreated or treated with 0.1 μg/mL TNF-α, 0.1 pM to 200 nMisotype control or 0.1 pM to 200 nM anti-BCMA antibodies for 20 min.Cells are then harvested, washed, and lysed with 50 mmol/L Tris-HCl (pH7.5), 1% NP0, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA supplementedwith protease, and phosphatase inhibitors. Protein extracts are thenanalyzed for NF-κB activity using a TransAM® chemiluminescent assay kit(Active Motif) and the luminescent signal reading is performed with aFusion HT plate reader (Packard Instruments). Anti-BCMA antibodies thatdo not induce downstream signaling and NF-κB activation (i.e. that themean luminescent signal from p65 bound to the NF-κB consensus sequencedetected by ELISA in nuclear extracts from NCI-H929 MM cells treatedwith anti-BCMA antibodies alone is similar, not significantly increased,to that of nuclear extracts from NCI-H929 MM cells treated with isotypecontrol antibody) are finally selected for further production and invitro and in vivo characterization. They represent the anti-BCMAantibodies that are non-ligand blocking, non-competing, andnon-signaling (see Table 10).

b) It was assessed whether binding of anti-BCMA antibodies toBCMA-expressing H929 cells induces NFkB activation, a known signalingpathway downstream of BCMA. Briefly, H929 cells were starved in RPMI1640 with 0.25% FCS for 24 h at 37° C. in cell incubator. At the end ofthe starvation time, cells were harvested, counted and cell viabilityevaluated using ViCell. Viable cells were adjusted to 4×10⁶ cells per mlin BSA-containing FACS Stain Buffer (BD Biosciences). 30 μl of this cellsuspension were further aliquoted per well into a round-bottom 96-wellplate and incubated with 30 μl of the anti-BCMA antibodies at 100 or 350nM (14 or 50 ug/ml) for 20 min at 37° C. As negative controls, cellswere either left untreated or incubated with the corresponding IgGisotype control antibodies 100 nM (14 ug/ml) for 20 min at 37° C. Aspositive controls, cells were incubated with 1 ug/ml recombinant mouseΔ-APRIL tagged with hemagglutinin (HA) (R&D Systems Europe,#7907-AP-010) for 20 min at 37° C. At the end of incubation time, cellswere harvested, washed, lysed, and processed according to themanufacturer's protocol of the Nuclear Extract Kit (Active Motif,#40410). Protein extracts were analyzed for NFkB activity using aTransAm© NFkB p65 Chemi Assay kit (Active Motif, #40097) followingmanufacturer's instructions. Luminescent signal was read using theSpectra Max M5 luminometer (Molecular Devices). The relativeluminescence signal intensity obtained from H929 cells treated asdescribed above was measured. The luminescence signal obtained uponbinding of Δ-APRIL in presence of the isotype control was set to one;the other signals were normalized to it.

TABLE 10 Screening paradigm for BCMA antibody selection SelectionDescription of Evaluation step (in chronologic order) criteriatechnique 1) Binding to BCMA Binding Binding to plate- bound-BCMA cells(ELISA) 2) Binding to BCMA not reduced by No Binding to plate- 100ng/mL, preferably 1000 ng/mL reduction bound-BCMA is APRIL or BAFF notaffected by 100 ng/mL, preferably 1000 ng/mL of APRIL or BAFF (ELISA) 3)Non-Activation of NF-κB No change APRIL/BAFF- downstream signaling inall three dependent 3.1) Does anti-BCMA antibody cases activation in MMblock or increase APRIL- cell line NCI-H929 dependent NF-κB activation?(chemiluminescent 3.2) Does anti-BCMA antibody ELISA) block or increaseBAFF- dependent NF-κB activation? 3.3) Does anti-BCMA antibody induceNF-κB activation by itself?

Example 2—Production of Therapeutic Anti-BCMA Antibodies that do notBlock Ligand (APRIL, BAFF) Binding and Neither Promote Nor BlockSignaling Via the BCMA Intracellular Domain and Whose Binding to BCMA isnot Affected by 100 ng/ml, Preferably 1000 ng/mL of APRIL or by 100ng/ml, Preferably 1000 ng/mL of BAFF

If the selected antibodies after step (Example 1H5) above are derivedfrom the in vitro selection out of the recombinant antibody library,then they are already unconjugated human IgG1 antibodies. Those selectedantibodies after step (Example 1H5) above that are derived fromimmunization are in a rat-human chimeric format and are then preferablyhumanized to be able to apply them for therapy. In that case, standardantibody humanization methods are applied by transferring thecomplementarity-determining regions of those rat variable regions intohuman antibody variable region frameworks. Additional mutations areintroduced into the variable regions, if necessary, to recover bindingto BCMA as compared to the chimeric, parental antibody.

For the production of the antibody, the cells are co-transfected withtwo plasmids, (one for expression of the heavy chain of the antibody andanother for expression of the light chain of the antibody), at a ratioof 1:1, respectively. Cells are grown as adherent monolayer cultures inT flasks using DMEM culture medium supplemented with 10% FCS, and aretransfected when they are between 50 and 80% confluent. For thetransfection of a T75 flask, 8 million cells are seeded 24 hours beforetransfection in 14 ml DMEM culture medium supplemented with FCS (at 10%V/V final), 250 μg/ml neomycin, and cells are placed at 37° C. in anincubator with a 5% CO₂ atmosphere overnight. For each T75 flask to betransfected, a solution of DNA, CaCl₂ and water is prepared by mixing 47μg total plasmid vector DNA divided equally between the light and heavychain expression vectors, 235 μl of a 1M CaCl₂ solution, and addingwater to a final volume of 469 μl. To this solution, 469 μl of a 50 mMHEPES, 280 mM NaCl, 1.5 mM Na₂HPO₄ solution at pH 7.05 are added, mixedimmediately for 10 sec and left to stand at room temperature for 20 sec.The suspension is diluted with 12 ml of DMEM supplemented with 2% FCS,and added to the T75 in place of the existing medium. The cells areincubated at 37° C., 5% CO₂ for about 17 to 20 hours, then medium isreplaced with 12 ml DMEM, 10% FCS. The conditioned culture medium isharvested 5 to 7 days post-transfection centrifuged for 5 min at 1200rpm, followed by a second centrifugation for 10 min at 4000 rpm and keptat 4° C.

The secreted antibodies are purified by Protein A affinitychromatography, followed by cation exchange chromatography and a finalsize exclusion chromatographic step on a Superdex® 200 column (AmershamPharmacia) exchanging the buffer to phosphate buffer saline andcollecting the pure monomeric IgG1 antibodies. Antibody concentration isestimated using a spectrophotometer from the absorbance at 280 nm. Theantibodies were formulated in a 25 mM potassium phosphate, 125 mM sodiumchloride, 100 mM glycine solution of pH 6.7.

Example 3—Generation of Anti-BCMA/Anti-CD3 T Cell Bispecific AntibodiesExample 3A. Generation of Anti-CD3 Antibodies

The following protein sequences of the VH and VL regions are used togenerate human and cynomolgus monkey cross reactive CD3E antibodies asdescribed in WO2007/042261.

H2C_VH (SEQ ID NO:7):

EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

H2C_VL (SEQ ID NO:8)

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVF GGGTKLTVL

Briefly, oligonucleotides encoding the above sequences are joinedtogether via PCR to synthesize cDNAs encoding the VH are VL sequences,respectively, of the anti-CD3 antibody.

Example 3B. Generation of Anti-BCMA/Anti-CD3 T Cell Bispecific 1+1Format with Fc

Anti-BCMA/anti-CD3 T cell bispecific are produced for the human orhumanized anti-BCMA antibodies selected after step (Example 1H5). cDNAsencoding the full heavy and light chains of the corresponding anti-BCMAIgG1 antibodies, as described in Example 2, as well as the anti-CD3 VHand VL cDNAs describe in Example 3A, are used as the starting materials.For each bispecific antibody, four protein chains are involvedcomprising the heavy and light chains of the corresponding anti-BCMAantibody and the heavy and light chains of the anti-CD3 antibodydescribed above, respectively. In order to minimize the formation ofside-products with mispaired heavy chains, for example with two heavychains of the anti-CD3 antibody, a mutated heterodimeric Fc region isused carrying “knob-into-hole mutations” and an engineered disulphidebond, as described in WO2009080251 and in WO2009080252. In order tominimize the formation of side-products with mispaired light chains, forexample with two light chains of the anti-BCMA antibody, a CH1× constantkappa crossover is applied to the heavy and light chains of the anti-CD3antibody using the methodology described in WO2009080251 and inWO2009080252.

Briefly, each bispecific antibody is produced by simultaneouscotransfection of four mammalian expression vectors encoding,respectively: a) the full light chain cDNA of the corresponding BCMAantibody, b) the full heavy chain cDNA of the corresponding BCMAantibody carrying the “hole mutations” in the Fc region to produce aheterodimeric antibody (see details below), c) a fusion cDNA generatedby standard molecular biology methods, such as splice-overlap-extensionPCR, encoding a fusion protein made of (in N- to C-terminal order)secretory leader sequence, VL of the anti-CD3 antibody described aboveand human CH1 domain of an IgG1 antibody and d) a fusion cDNA generatedby standard molecular biology methods, such as splice-overlap-extensionPC, encoding a fusion protein made of (in N- to C-terminal order)secretory leader sequence, VH of the anti-CD3 antibody described above,constant kappa domain of a human light chain cDNA, hinge region of ahuman IgG1 antibody, and Fc region (CH2 and CH3 domains) of a human IgG1antibody including a “knob mutation” (see details below) in the Fcregion to produce a heterodimeric antibody. Co-transfection of mammaliancells and antibody production and purification using the methodsdescribed above for production of human or humanized IgG1 antibodies(see Example 2). The “knob-into-hole mutations” in the human IgG1 Fcregion consist of: T366W, known as the “knob mutation”; and T366S,L368A, and Y407V, collectively known as the “hole mutations”. Inaddition, a disulfide can be included to increase the stability andyields as well as additional residues forming ionic bridges andincreasing the heterodimerization yields (EP 1870459A1).

Example 4—Simultaneous Binding of Anti-BCMA/Anti-CD3 T Cell BispecificAntibodies to BCMA and CD3 (Surface Plasmon Resonance)

The binding properties to BCMA and CD3 of bispecific anti-BCMA/anti-CD3T cell bispecific antibodies generated in Example 3 are analyzed bysurface plasmon resonance (SPR) technology using a Biacore® T100instrument (Biacore AB) with HBS-EP as running buffer (0.01 M HEPES pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore). Thissystem is well established for the study of molecule interactions. Itallows a continuous real-time monitoring of ligand/analyte bindings andthus the determination of association rate constants (ka), dissociationrate constants (kd), and equilibrium constants (KD) in various assaysettings. SPR-technology is based on the measurement of the refractiveindex close to the surface of a gold coated biosensor chip. Changes inthe refractive index indicate mass changes on the surface caused by theinteraction of immobilized ligand with analyte injected in solution. Ifmolecules bind to immobilized ligand on the surface the mass increases,in case of dissociation the mass decreases.

Capturing anti-His tag antibody is immobilized on the surface of a CM5biosensorchip using amine-coupling chemistry. Flow cells are activatedwith a 1:1 mixture of 0.1 M N-hydroxysuccinimide and 0.1 M3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide at a flow rate of 5μl/min anti-human IgG antibody is injected in sodium acetate, pH 5.0 at10 μg/ml, which resulted in a surface density of approximately 12000resonance units (RU). A reference control flow cell is treated in thesame way but with vehicle buffers only instead of the capturingantibody. Surfaces are blocked with an injection of 1 M ethanolamine/HClpH 8.5. The anti-BCMA/anti-CD3 T cell bispecific antibodies are dilutedin HBS-P and injected at a flow rate of 5 μl/min. The contact time(association phase) is 1 min for the antibodies at a concentrationbetween 1 and 100 nM for the BCMA-ECD binding and 1 and 200 nM for theCD3 interaction. BCMA-ECD is injected at increasing concentrations of3.125, 6.25, 12.5, 25, 50 and 100 nM, CD3 at concentrations of 0.21,0.62, 1.85, 5.6, 16.7, 50, 100 and 200 nM. The contact time (associationphase) is 3 min, the dissociation time (washing with running buffer) 5min for both molecules at a flowrate of 30 μl/min. All interactions areperformed at 25° C. (standard temperature). The regeneration solution of3 M Magnesium chloride is injected for 60 s at 5 μl/min flow to removeany non-covalently bound protein after each binding cycle. Signals aredetected at a rate of one signal per second. Samples are injected atincreasing concentrations. SPR graphs showing the rate of signal (i.e.resonance unit) plotted against contact time are determined.

Example 5—Binding of Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies toBCMA on MM Cells or CD3 on T Cells (Flow Cytometry)

Anti-BCMA/anti-CD3 T cell bispecific antibodies generated in Example 3are also analyzed by flow cytometry for their binding properties tohuman BCMA expressed on NCI-H929 multiple myeloma cells or human CD3expressed on human leukemic T cells Jurkat (ATCC). Briefly, culturedcells are harvested, counted and cell viability is evaluated using theTrypan Blue exclusion method. Viable cells are then adjusted to 2×10⁶cells per ml in PBS containing 0.1% BSA. 90 μl of this cell suspensionare further aliquoted per well into a round-bottom 96-well plate. 10 μlof the T cell bispecific antibody or corresponding IgG control are addedto the cell-containing wells to obtain final concentrations of 0.1 pM to200 nM. Anti-BCMA/anti-CD3 T cell bispecific antibodies and control IgGare used at the same molarity. After incubation for 30 min at 4° C.,cells are centrifuged (5 min, 350×g), washed with 150 μl/wellBSA-containing FACS Stain Buffer (BD Biosciences), resuspended andincubated for an additional 30 min at 4° C. with 12 μl/wellfluorochrome-conjugated anti-His antibody (Lucerna) for detection of theT cell bispecific antibody. Cells are then washed by addition of 120μl/well FACS Stain Buffer and centrifugation at 350×g for 5 min. Asecond washing step is performed with 150 μl/well FACS Stain Buffer. Thesamples are resuspended in 200 μl/well FACS Stain Buffer, acquired andanalyzed using an LSR II flow cytometer with FACSDiva® software (BDBiosciences). Binding of the anti-BCMA/anti-CD3 T cell bispecificantibodies to MM cells and T cells are evaluated and the meanfluorescence intensity is determined gated on either BCMA-expressingNCI-H929 MM cells or CD3-expressing Jurkat T cells and plotted inhistograms or dot plots.

Example 6—Activation of T Cells Upon Engagement of Anti-BCMA/Anti-CD3 TCell Bispecific Antibodies (Flow Cytometry)

Anti-BCMA/anti-CD3 T cell bispecific antibodies generated in Example 3are also analyzed by flow cytometry for their potential to induce T cellactivation by evaluating the surface expression of the early activationmarker CD69, or the late activation marker CD25 on CD4⁺ and CD8⁺ T cellsin the presence or absence of human BCMA-expressing MM cells. Briefly,BCMA-expressing NCI-H929 MM cells are harvested with Cell Dissociationbuffer, counted and cell viability is verified using Trypan Blue. ViableMM cells are adjusted to 0.2×10⁶ cells/mL in complete RPMI-1640 medium,100 μl of this cell suspension per well is pipetted into a round-bottom96-well plate. 50 μl of the T cell bispecific constructs are added tothe MM cells-containing wells to obtain a final concentration of 1 nM.The 96-well plate is set aside and kept at 37° C., 5% CO₂ until furthermanipulations.

PBMC are isolated from fresh blood using density gradient centrifugationusing Cell Preparation Tubes with Sodium citrate (Vacutainer CPT tubes,BD Biosciences). Total human T cells are then isolated using the Pan TCell Isolation Kit II (Miltenyi Biotec), according to the manufacturer'sinstructions. Human total T cells (effector) are then adjusted to 2×10⁶cells per ml in complete RPMI-1640 medium. 50 μl of this cell suspensionis added per well in the assay plate containing already BCMA-expressingMM cells to obtain a final E:T ratio of 5:1. To test whether the T cellbispecific constructs are able to activate T cells only in the presenceof BCMA-expressing MM tumor target cells, wells containing finalconcentration(s) in the range of 0.1 pM to 200 nM of the respectivebispecific molecules with effector cells but without MM tumor targetcells are also included. After incubation for five days at 37° C., 5%CO₂, cells are pelleted down by centrifugation (5 min, 350×g) and washedtwice with 150 μl/well of FACS Stain Buffer (BD Biosciences). Surfacestaining of the effector cells with selected fluorochrome-conjugatedantibodies against human CD4, CD8, CD69 or CD25 (BD Biosciences) isperformed at 4° C. for 30 min, protected from light, in FACS StainBuffer (BD Biosciences) according to the manufacturer's protocol. Cellsare washed twice with 150 μl/well FACS Stain Buffer, resuspended in 200μl/well FACS Stain Buffer, and acquired and analyzed using a LSRII flowcytometer complemented with FACSDiva® software (BD Biosciences). Theexpression of CD69 and CD25 activation markers are determined bymeasuring the mean fluorescence intensity gated on CD4⁺ and CD8⁺ T cellpopulations as represented in histograms or dot plots.

Example 7—Proliferation of T Cells Upon Engagement of Anti-BCMA/Anti-CD3T Cell Bispecific Antibodies (CFSE Dilution)

Anti-BCMA/anti-CD3 T cell bispecific antibodies generated in Example 3are also analyzed by flow cytometry for their potential to induceproliferation of CD8⁺ or CD4⁺ T cells in the presence or absence ofhuman BCMA-expressing MM cells. Briefly, BCMA-expressing NCI-H929 MMcells are harvested with Cell Dissociation buffer, counted and lookedfor viability using Trypan Blue. Viable MM cells are adjusted to 0.2×10⁶cells per ml in complete RPMI medium, 100 μl of this cell suspension arepipetted per well into a round-bottom 96-well plate. 50 μl of the T cellbispecific constructs are added to the MM cell-containing wells toobtain final concentration(s) in the range of 0.1 pM to 200 nM. The wellplate is set aside and kept at 37° C., 5% CO₂.

PBMC are isolated from fresh blood using density gradient centrifugationusing Cell Preparation Tubes with Sodium citrate (Vacutainer CPT tubes,BD Biosciences). Total human T cells are then isolated using the Pan TCell Isolation Kit II (Miltenyi Biotec), according to the manufacturer'sinstructions. The total T cells are then adjusted to 1 million cells perml in pre-warm RPMI without serum (37° C.) and stained with 1 μM CFSE atroom temperature for 6 min, protected from light. The staining volume isthen doubled by addition of RPMI-1640 medium supplemented with 10% FCSand 1% GlutaMax to stop CFSE staining. After incubation at roomtemperature for further 20 min, the cells are washed three times withpre-warmed serum-containing medium to remove remaining CFSE.CFSE-stained total T cells (effector) are then adjusted to 2×10⁶cells/mL in complete RPMI-1640 medium. 50 μl of this cell suspension isadded per well in the assay plate already containing BCMA-expressingNCI-H929 MM cells to obtain a final E:T ratio of 5:1. To test whetherthe T cell bispecific constructs are able to activate T cells only inthe presence of BCMA-expressing MM tumor target cells, wells containing1 nM of the T cell bispecific antibodies with effector cells but withoutMM tumor target cells are also included. After incubation for five daysat 37° C., 5% CO₂, cells are pelleted down by centrifugation (5 min,350×g) and washed twice with 150 μl/well of FACS Stain Buffer (BDBiosciences). Surface staining of the effector cells with selectedfluorochrome-conjugated antibodies against human CD4, CD8 or CD25 (BD)is performed at 4° C. for 30 min, protected from light, in FACS StainBuffer according to the manufacturer's protocol. Cells are washed twicewith 150 μl/well FACS Stain Buffer, resuspended in 200 μl/well FACSStain Buffer, and acquired and analyzed using a LSR II flow cytometercomplemented with FACSDiva® software (BD). The percentage ofnon-proliferating cells is determined by gating on the far rightundiluted CFSE peak in the group which the wells contain BCMA-expressingMM cells and CFSE-stained T cells but without the T cell bispecificantibodies, and compared that to other experimental groups (wells). Thepercentage of proliferating cells is measured by gating all the dilutedCFSE peaks excluding the far right peak (if observable). Theproliferation level of CD4⁺ and CD8⁺ T cells is determined by gating onthat population first then to further look at the CFSE dilution peaks.

Example 8—Cytokine Production from Activated T Cells Upon Engagement ofAnti-BCMA/Anti-CD3 T Cell Bispecific Antibodies Example 8A. Interferon-γProduction

Anti-BCMA/anti-CD3 T cell bispecific antibodies generated in Example 3are also analyzed for their potential to induce interferon-γ (IFN-γ)production by the T cells in the presence or absence of humanBCMA-expressing MM cells. Briefly, BCMA-expressing NCI-H929 MM cells areharvested with Cell Dissociation buffer, counted and looked forviability using Trypan Blue. Approximately 20,000 viable cells per wellare plated in a round-bottom 96-well-plate and the respective antibodydilution is added to obtain final concentration(s) in the range of 0.1pM to 200 nM. Anti-human BCMA and anti-CD3 IgGs adjusted to the samemolarity are used as controls. Human total T effector cells are added toobtain a final E:T ratio of 5:1. After 20 h incubation at 37° C., 5%CO₂, human IFN-γ levels in the supernatant are measured by ELISA,according to the manufacturer's instructions (human IFN-γ ELISA Kit II,BD Biosciences). The levels of IFN-γ produced by T cells in the presenceof anti-BCMA/anti-CD3 T cell bispecific antibody and BCMA-expressing MMcells is measured and plotted in histograms and compared to thatproduced by T cells in the presence of anti-BCMA/anti-CD3 T cellbispecific antibody and but without BCMA-expressing MM cells.

Example 8B. Cytokine Release Assay (CBA Analysis)

Anti-BCMA/anti-CD3 T cell bispecific antibodies generated in Example 3are also analyzed for their potential to induce T-cell mediated cytokineproduction in the presence or absence of human BCMA-expressing MM cells.PBMC are isolated from fresh blood using density gradient centrifugationusing Cell Preparation Tubes with Sodium citrate (Vacutainer CPT tubes,BD Biosciences) and a final cell concentration of 0.3 million cells/wellare plated into a round-bottom 96-well plate. BCMA-expressing NCI-H929MM cells are then added to obtain a final E:T-ratio of 10:1, as well asT cell bispecific constructs and IgG controls are added to obtain finalconcentration(s) in the range of 0.1 pM to 200 nM, for a 24 h incubationat 37° C., 5% CO₂. The next day, the cells are centrifuged for 5 min at350×g and the supernatant is transferred into a new deep-well96-well-plate for the further analysis. The CBA analysis is performedaccording to manufacturer's instructions for ISR II flow cytometer,using the Human Th1/Th2 Cytokine Kit II (BD Biosciences) including humanIL-2, human IL-4, human IL-6, human IL-10, human TNF-α, and human IFN-γ.The levels of cytokines produced by T cells in the presence ofanti-BCMA/anti-CD3 T cell bispecific antibody and BCMA-expressing MMcells is measured and plotted in histograms and compared to thatproduced by T cells in the presence of anti-BCMA/anti-CD3 T cellbispecific antibody and but without BCMA-expressing MM cells.

Example 9—Redirected T Cell Cytotoxicity of MM Cells Upon Cross-Linkingof Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies to CD3 on T Cells andBCMA on MM Cells (LDH Release Assay)

Anti-BCMA/anti-CD3 T cell bispecific antibodies generated in Example 3are also analyzed for their potential to induce T cell-mediatedapoptosis in BCMA-expressing MM cells upon crosslinking of the constructvia binding of the antigen binding moieties to BCMA on cells. Briefly,human BCMA-expressing NCI-H929 multiple myeloma target cells areharvested with Cell Dissociation Buffer, washed and resuspended in RPMIsupplemented with 10% fetal bovine serum (Invitrogen). Approximately,30,000 cells per well are plated in a round-bottom 96-well plate and therespective dilution of the construct is added for a desired finalconcentration (in triplicates); final concentrations ranging from 0.1 pMto 200 nM. For an appropriate comparison, all T cell bispecificconstructs and controls are adjusted to the same molarity. Human total Tcells (effector) are added into the wells to obtain a final E:T ratio of5:1. When human PBMC are used as effector cells, a final E:T ratio of10:1 is used. PHA-L (Sigma) is used as positive control for human T cellactivation at a concentration of 1 μg/ml. Negative control groups arerepresented by effector or target cells only. For normalization, maximallysis of the NCI-H929 MM target cells (=100%) is determined byincubation of the target cells with a final concentration of 1% TritonX-100, inducing cell death. Minimal lysis (=0%) is represented by targetcells co-incubated with effector cells only, i.e. without any T cellbispecific antibody. After 20 h incubation at 37° C., 5% CO₂, LDHrelease from the apoptotic/necrotic MM target cells into the supernatantis then measured with the LDH detection kit (Roche Applied Science),following the manufacturer's instructions. The percentage of LDH releaseis plotted against the concentrations of anti-BCMA/anti-CD3 T cellbispecific antibodies in concentration-response curves. The IC₅₀ valuesare measured using Prism software (GraphPad) and determined as the Tcell bispecific antibody concentration that results in 50% of LDHrelease.

Example 10—Comparison of T Cell Bispecifics Containing a Non-LigandBlocking/Non-Competing Anti-BCMA Antibody vs. aLigand-Blocking/Competing Anti-BCMA Antibody on the Killing Potency ofBCMA-Expressing MM Cells

In certain hematological malignancies such as multiple myeloma, thelevel of circulating BCMA-ligands APRIL and BAFF can be elevated(Moreaux et al. 2004; Blood 103(8): 3148-3157). Thus, the inventorsrecognize that high levels of ligands in the serum may interfere withthe binding of anti-BCMA antibodies to BCMA on the tumor cells. Incomparison to healthy donors, the levels of circulating APRIL (the highaffinity ligand to BCMA) in MM patient are ˜100 ng/mL vs. ˜10 ng/mL. ForBAFF (the low affinity ligand to BCMA), the levels can fluctuate from1-1000 ng/mL as compared to ˜3 ng/mL in healthy donors. Close to thetumor cells APRIL/BAFF concentrations may be well even higher thanmeasured in the serum. In certain autoimmune diseases such as systemiclupus erythematosus, the levels of circulating APRIL are also elevatedwith ˜85 ng/mL (Koyama et al. 2005; Ann Rheum Dis 64: 1065-1067).

Anti-BCMA/anti-CD3 T cell bispecific antibodies generated in Example 3containing a non-ligand blocking/non-competing anti-BCMA antibody arealso analyzed for their potential to induce T cell-mediated apoptosis inBCMA-expressing MM cells upon crosslinkage of the construct via bindingof the antigen binding moieties to BCMA on cells in the presence ofelevated concentrations (i.e. 100 ng/mL to 1000 ng/mL) of APRIL or BAFFin comparison to anti-BCMA/anti-CD3 T cell bispecific antibodiescontaining a ligand blocking/competing anti-BCMA antibody of the sameformat.

As shown in FIG. 1, the increasing concentrations (i.e. 10, 100, 1000ng/mL) of soluble APRIL or BAFF representative of the levels found inthe blood and bone marrow of multiple myeloma patients do not alter thebinding of a non-ligand blocking/non-competing anti-BCMA antibody toplate-bound-BCMA (continuous line). In contrast, the high concentrationsof soluble APRIL or BAFF representative of the levels (i.e. 100 ng/mL to1000 ng/mL) found in the blood and bone marrow of multiple myelomapatients decrease the binding of a ligand blocking/competing anti-BCMAantibody to plate-bound-BCMA (dotted line).

As shown in FIG. 2, the increasing concentrations (i.e. 10, 100, 1000ng/mL) of soluble APRIL or BAFF representative of the levels found inthe blood and bone marrow of multiple myeloma patients do not alter thekilling potency of a T cell bispecific antibody containing a non-ligandblocking/non-competing anti-BCMA antibody specific to BCMA-expressing MMcells (continuous line). In contrast, the high concentrations (i.e. 100ng/mL to 1000 ng/mL) of soluble APRIL or BAFF representative of thelevels found in the blood and bone marrow of multiple myeloma patientsdecrease the killing potency of a T cell bispecific antibody containinga ligand blocking/competing anti-BCMA antibody specific toBCMA-expressing MM cells (dotted line).

Example 10A. Binding Properties of Anti-BCMA/Anti-CD3 T Cell BispecificAntibodies to BCMA-Expressing MM Cells with a Non-LigandBinding/Blocking, Non-Competing Anti-BCMA Antibody in the Presence of10, 100, 1000 ng/mL of APRIL or BAFF (Flow Cytometry)

Anti-BCMA/anti-CD3 T cell bispecific antibodies with a non-ligandbinding/blocking, non-competing anti-BCMA antibody generated in Example3 are analyzed by flow cytometry for their binding properties to humanBCMA expressed on NCI-H929 multiple myeloma cells in the presence of 10,100 and 1000 ng/mL of APRIL or BAFF. Briefly, cultured cells areharvested, counted and cell viability is evaluated using the Trypan Blueexclusion method. Viable cells are then adjusted to 2×10⁶ cells per mlin PBS containing 0.1% BSA. 90 μl of this cell suspension are furtheraliquoted per well into a round-bottom 96-well plate. 10 μl of the Tcell bispecific antibody or corresponding IgG control are added to thecell-containing wells to obtain preferably final concentrations rangingfrom 0.1 pM to 200 nM. Anti-BCMA/anti-CD3 T cell bispecific antibodiesand control IgG are used at the same molarity. After incubation for 30min at 4° C., cells are centrifuged (5 min, 350×g), washed with 150μl/well BSA-containing FACS Stain Buffer (BD Biosciences), resuspendedand incubated for an additional 30 min at 4° C. with 12 μl/wellfluorochrome-conjugated anti-His antibody (Lucerna) for detection of theT cell bispecific antibody. Cells are then washed by addition of 120μl/well FACS Stain Buffer and centrifugation at 350×g for 5 min. Asecond washing step is performed with 150 μl/well FACS Stain Buffer. Thesamples are resuspended in 200 μl/well FACS Stain Buffer, acquired andanalyzed using an LSR II flow cytometer with FACSDiva® software (BDBiosciences). Binding of the anti-BCMA/anti-CD3 T cell bispecificantibodies to MM cells and T cells are evaluated and the meanfluorescence intensity is determined gated on BCMA-expressing NCI-H929MM cells and plotted in histograms or dot plots. The binding (e.g. MFI)of an anti-BCMA/anti-CD3 T cell bispecific antibodies with a non-ligandbinding/blocking, non-competing anti-BCMA antibody to MM cells is thencompared to that of an anti-BCMA/anti-CD3 T cell bispecific antibodieswith a ligand binding/blocking, competing anti-BCMA antibody in thepresence of 0, 10, 100, 1000 ng/mL of APRIL or BAFF.

Example 10B. Killing Properties of Anti-BCMA/Anti-CD3 T Cell BispecificAntibodies with a Non-Ligand Binding/Blocking, Non-Competing Anti-BCMAAntibody in the Presence of 10, 100, 1000 Ng/mL of APRIL or BAFF:Redirected T Cell Cytotoxicity of BCMA-Expressing MM Cells (LDH ReleaseAssay)

Anti-BCMA/anti-CD3 T cell bispecific antibodies with a non-ligandbinding/blocking, non-competing anti-BCMA antibody generated in Example3 are analyzed for their potential to induce T cell-mediated apoptosisin BCMA-expressing MM cells upon crosslinking of the construct viabinding of the antigen binding moieties to BCMA on cells, in thepresence or absence of increasing concentrations (i.e. 10, 100, 1000ng/mL) of APRIL or BAFF. Briefly, human BCMA-expressing NCI-H929multiple myeloma target cells are harvested with Cell DissociationBuffer, washed and resuspended in RPMI supplemented with 10% fetalbovine serum (Invitrogen). Approximately, 30,000 cells per well areplated in a round-bottom 96-well plate and the respective dilution ofthe T cell bispecific antibody is added preferably for concentration(s)in the range of 0.1 pM to 200 nM (in triplicates). For an appropriatecomparison, all T cell bispecific antibodies and controls are adjustedto the same molarity. Increasing concentrations (i.e. 10, 100, 1000ng/mL) of soluble human recombinant APRIL or BAFF are added to the cellcultures. Wells without addition of APRIL or BAFF are also included inthe plate as controls. Human total T cells (effector) are then addedinto the wells to obtain a final E:T ratio of 5:1. When human PBMC areused as effector cells, a final E:T ratio of 10:1 is used. PHA-L (Sigma)is used as positive control for human T cell activation at aconcentration of 1 μg/ml. Negative control groups are represented byeffector or target cells only. For normalization, maximal lysis of theNCI-H929 MM target cells (=100%) is determined by incubation of thetarget cells with a final concentration of 1% Triton X-100, inducingcell death. Minimal lysis (=0%) is represented by target cellsco-incubated with effector cells only, i.e. without any construct orantibody. After 20 h incubation at 37° C., 5% CO₂, LDH release from theapoptotic/necrotic MM target cells into the supernatant is then measuredwith the LDH detection kit (Roche Applied Science), following themanufacturer's instructions. The percentage of LDH release is plottedagainst the concentrations of APRIL or BAFF in the presence ofconcentration(s) of anti-BCMA/anti-CD3 T cell bispecific antibodiespreferably in the concentration range of 0.1 pM to 200 nM inconcentration-response curves. The IC₅₀ values are then measured usingPrism software (GraphPad). The IC₅₀ values of an anti-BCMA/anti-CD3 Tcell bispecific antibodies with a non-ligand binding/blocking,non-competing anti-BCMA antibody is then compared to that of ananti-BCMA/anti-CD3 T cell bispecific antibodies with a ligandbinding/blocking, competing anti-BCMA antibody in the presence of 0, 10,100, 1000 ng/mL of APRIL or BAFF.

Example 11—Evaluation of Therapeutic Efficacy of Anti-BCMA/Anti-CD3 TCell Bispecific Antibody in the Vk*MYC Multiple Myeloma Mouse Model

Murine cross-reactive anti-BCMA/anti-CD3 T cell bispecific antibodiesare tested for their potential to prevent multiple myeloma in Vk*MYCmultiple myeloma prone mice as described in Chesi, 2012 (Chesi et al.2012; Blood 120: 376-385). Multiple myeloma is a hematologicalmalignancy involving an uncontrolled expansion of plasma cells in thebone marrow. Since BCMA is strongly expressed on malignant plasma cells,we hypothesize that an anti-BCMA/anti-CD3 T cell bispecific will beefficacious for the treatment of multiple myeloma. The Vk*MYC multiplemyeloma mouse model is highly representative of human myeloma andpredictive of drug response used in the clinic; representing anexcellent tool for testing the preclinical proof-of-concept ofanti-BCMA/anti-CD3 T cell bispecific antibodies. Briefly, Vk*MYC miceobtained from an academic collaboration at Mayo Clinic Arizona arecrossed with human CD3E transgenic (huCD3ε Tg) mice. T cells from huCD3εTg×Vk*MYC mice express both human CD3E and mouse CD3E on the cellsurface and the mice are therefore responsive to anti-BCMA/anti-CD3 Tcell bispecifics. Vk*MYC mice uniformly develop a monoclonal gammopathyinitiated at around 30 weeks of age that progresses slowly over timeassociated with clinical signs representative of human myeloma such asanemia, osteoporosis and renal disease. Mice are periodically bled bytail grazing and blood is collected into Microtainer tubes (BDBiosciences), let coagulate at room temperature then spun for 10 min at2,300 g. Sera are diluted 1:2 in normal saline buffer and analyzed on aQuickGel Chamber apparatus using pre-casted QuickGels (HelenaLaboratories) according to manufacturer's instruction. Gamma/albuminratio and serum fractions are measured by densitometric analysis.

For therapeutic studies, Vk*MYC mice are enrolled and randomized intodifferent treatment groups (n=5-8/group): for example, 1) control IgGs;2) anti-BCMA/anti-CD3 T cell bispecific antibodies; 500 Kg/kg/week or 10Kg/mouse/week administered intravenously via the tail vein; 3)bortezomib 1 mg/kg/i.p. on days 1, 4, 8, 11 used as standard of care.Preferably, the dose(s) of anti-BCMA/anti-CD3 T cell bispecificantibodies could be multiple and range from 200 to 1000 Kg/kg/week. Ineach group, at least three aged (>1 year old) Vk*MYC mice withgamma/albumin ratio between 0.3-2.0, corresponding to a predominantM-spike between approximately 10-70 g/l as measured by densitometry.Serum protein electrophoresis (SPEP) is performed on day 0 and day 14post treatment to measure treatment-mediated reduction in the M-spike asa marker of tumor response, as done in the clinic. In some therapeuticstudies, transplanted Vk*MYC mice with an M-spike approximately 10-70g/l and a bone marrow plasmacytosis of greater than 5% are enrolled andassigned to different treatment groups. The efficacy ofanti-BCMA/anti-CD3 T cell bispecific antibodies to reduce M-spike isevaluated.

Example 12—Evaluation of Therapeutic Efficacy in the NZB/W Lupus ProneMouse Model of Systemic Lupus Erythematosus

Murine cross-reactive anti-BCMA/anti-CD3 T cell bispecific antibodiesare tested for their potential to prevent systemic lupus erythematosus(SLE) in the NZB/W prone mice, a well-characterized model (Hass et al.2010; J Immunol 184(9): 4789-4800). There is accumulating evidencesuggesting that autoreactive plasma cells play an important role in SLEand depletion of the autoreactive plasma cells with ananti-BCMA/anti-CD3 T cell bispecific could be beneficial to SLEpatients. Briefly, NZB and NZW mice are purchased from the JacksonLaboratory and crossed with huCD3ε Tg mice. NZB×huCD3ε Tg mice andNZW×huCD3ε Tg mice are then crossed with each other and female huCD3εTg×NZB/W F1 mice are selected for future studies. Mice are testedsemiquantitatively for proteinuria with Albustix reagent strips (SiemensHealthcare Diagnostics Inc.) every other week, and scored on a scalefrom 0 to 4 according to protein concentration (from 0 to ≥20 g/l).huCD3ε Tg×NZB/W F1 female mice of 7-8 months of age are enrolled intherapeutic studies and randomized into different treatment groups(n=16/group): for example, 1) control IgGs; 2) anti-BCMA/anti-CD3 T cellbispecific antibodies; 500 μg/kg/week or 10 Kg/mouse/week administeredintravenously via the tail vein; 3) anti-BAFF 20 mg/kg/week used asstandard of care. Preferably, the dose(s) of anti-BCMA/anti-CD3 T cellbispecific antibodies could be multiple and range from 200 to 1000μg/kg/week. The baseline protein levels at the start of the therapeuticstudies are between 30 and 300 mg/dL.

The clinical endpoints representative of SLE consist of proteinuria,kidney diseases and manifestations such as glomerulonephritis,glomerular cellularity and size increase, periodic acid-Schiff(PAS)-positive deposits, and appearance of autoantibodies in sera suchas dsDNA, total IgA, IgG and IgM as measured by ELISA.

The invention claimed is:
 1. A bispecific antibody, in the format FabBCMA-Fc-Fab CD3-Fab BCMA, specifically binding to the two targets humanCD3ε and human BCMA (B-cell maturation antigen), wherein said bispecificantibody comprises (a) a first Fab fragment of an antibody specificallybinding to human BCMA (first Fab fragment of the anti-BCMA antibody);(b) a second Fab fragment of the antibody specifically binding to humanBCMA (second Fab fragment of the anti-BCMA antibody); (c) an Fc part;(d) a Fab fragment of an antibody specifically binding to human CD3ε(Fab fragment of the anti-CD3ε antibody), wherein the variable domainsVL and VH or the constant domains CL and CH1 of the Fab fragment of theanti-CD3ε antibody are replaced by each other; and, wherein the firstFab fragment of the anti-BCMA antibody is linked via its C-terminus tothe N-terminus of the hinge region of the Fc part, the Fab fragment ofthe anti-CD3ε antibody is linked via its C-terminus to the N-terminus ofthe hinge region of the Fc part, and the second Fab fragment of theanti-BCMA antibody is chemically linked via its C-terminus to theN-terminus of the Fab fragment of the anti-CD3ε antibody.
 2. A host cellcomprising vectors comprising nucleic acid molecules encoding lightchains and heavy chains of a bispecific antibody according to claim 1.3. A pharmaceutical composition comprising a bispecific antibodyaccording to claim 1 and a pharmaceutically acceptable excipient.
 4. Amethod of treating multiple myeloma comprising administering thebispecific antibody according to claim 1 to a subject in need thereof.5. The bispecific antibody according to claim 1, wherein the Fc part isan Fc variant of a wild-type human IgG1 Fc region and wherein the Fcvariant comprises an amino acid substitution at position Pro329 withglycine or arginine and at least one further amino acid substitution atposition L234A or L235A.